Microvolume crystallization method employing multiple lumens

ABSTRACT

A method for determining crystallization conditions for a material, the method comprising: taking a microfluidic device comprising one or more lumens having microvolume dimensions and a plurality of different crystallization samples within the one or more lumens, the plurality of crystallization samples comprising a material to be crystallized and crystallization conditions that vary among the plurality of crystallization samples; transporting the plurality of different crystallization samples within the lumens; and identifying a precipitate or crystal formed in the one or more lumens.

RELATED APPLICATION

This application is a continuation in part of U.S. patent applicationSer. No. 09/877,405 filed Jun. 8, 2001, now U.S. Pat. No. 6,719,840which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to microfluidic devices and methods.

2. Description of Related Art

Traditional methods for crystal growth and crystallization are highlylabor intensive and require significant quantities of material toevaluate and optimize crystal growth conditions. Examples of thesemethods include the free interface diffusion method (Salemme, F. R.(1972) Arch. Biochem. Biophys. 151:533-539), vapor diffusion in thehanging or sitting drop method (McPherson, A. (1982) Preparation andAnalysis of Protein Crystals, John Wiley and Son, New York, pp 82-127),and liquid dialysis (Bailey, K. (1940) Nature 145:934-935).

Presently, the hanging drop method is the most commonly used method forgrowing macromolecular crystals from solution, especially for proteincrystals. Generally, a droplet containing a protein solution is spottedon a cover slip and suspended in a sealed chamber that contains areservoir with a higher concentration of precipitating agent. Over time,the solution in the droplet equilibrates with the reservoir by diffusingwater vapor from the droplet, thereby slowly increasing theconcentration of the protein and precipitating agent within the droplet,which in turn results in precipitation or crystallization of theprotein.

The process of growing crystals with high diffraction quality istime-consuming and involves trial-and-error experiment on multiplesolution variables such as pH, temperature, ionic strength, and specificconcentrations of salts, organic additives, and detergents. In addition,the amount of highly purified protein is usually limited,multi-dimensional trials on these solution conditions are unrealistic,labor-intensive and costly.

A few automated crystallization systems have been developed based on thehanging drop methods, for example Cox, M. J. and Weber, P. C. (1987) J.Appl. Cryst. 20:366; and Ward, K. B. et al. (1988) J. Crystal Growth90:325-339. More recently, systems for crystallizing proteins insubmicroliter drop volumes have been described including those describedin PCT Publication Nos. WO00/078445 and WO00/060345.

Existing crystallization, such as hanging drop, sitting drop, dialysisand other vapor diffusion methods have the limitation that the materialfor analysis and the crystallization medium are exposed to theenvironment for some time. As the volumes of materials decrease, theratio of surface area to volume ratio varies as the inverse of theradius of the drop. This causes smaller volumes to be more susceptibleto evaporation during the initial creation of the correct mixture andduring the initial period after the volume has been set up. Typicalhanging drop plates can have air volumes of 1.5 milliliters compared toa sample drop size of 3-10 microliters. Moreover, typical methods exposethe sample drop to the environment for a duration of seconds to minutes.Small variability in the rate that samples are made can causesignificant variations in the production of crystals. Small variationsexternal environment also can cause significant variations in theproduction of crystals even if the rate that the samples are made isunchanged. Prior methods fail to reduce the problems of convectioncurrents under 1 g such as those described in U.S. Pat. No. 4,886,646,without the large expenditure of resources or in methods that complicatecrystal analysis.

SUMMARY OF THE INVENTION

The present invention relates to various microfluidics devices, methods,and kits.

In one embodiment, a microfluidic device is provided that comprises: acard shaped substrate having first and second opposing faces; one ormore microvolumes at least partially defined by a first face of the cardshaped substrate; and one or more grooves at least partially defined bya second face of the card shaped substrate; wherein a lateral footprintof at least a portion of the one or more grooves overlaps with a lateralfootprint of at least one of the one or more microvolumes.

Optionally, the one or more grooves are sufficiently deep relative tothe second face of the substrate within the overlapping lateralfootprint that when the portion of the microvolume within theoverlapping lateral footprint comprises a crystallization sample and anx-ray beam traverses the card shaped substrate at the overlappinglateral footprint, the portion of the microvolume that the x-ray beamtraverses contains at least half as many electrons as is contained inthe substrate where the x-ray beam traverses. Optionally, the portion ofthe microvolume that the x-ray beam traverses contains at least as manyelectrons as is contained in the substrate where the x-ray beamtraverses. Preferably, the portion of the microvolume that the x-raybeam traverses contains at least three, five, ten times or more times asmany electrons as is contained in the substrate where the x-ray beamtraverses.

Optionally, the one or more microvolumes comprise at least one lumen. Insuch an instance, the groove may have a longitudinal axis that isaligned with a longitudinal axis of the lumen adjacent the overlappinglateral footprint. The groove may also have a longitudinal axis that isperpendicular to a longitudinal axis of the lumen adjacent theoverlapping lateral footprint.

In another embodiment, a microfluidic device is provided that comprises:a card shaped substrate having first and second opposing faces; aplurality of microvolumes at least partially defined by a first face ofthe card shaped substrate; and one or more grooves at least partiallydefined by a second face of the card shaped substrate; wherein a lateralfootprint of at least a portion of the one or more grooves overlaps withlateral footprints of plurality of microvolumes.

In another embodiment, a method is provided for use with a microfluidicdevice, the method comprising: performing an experiment in amicrofluidic device comprising a card shaped substrate having first andsecond opposing faces, one or more microvolumes at least partiallydefined by a first face of the card shaped substrate; and one or moregrooves at least partially defined by a second face of the card shapedsubstrate; wherein a lateral footprint of at least a portion of the oneor more grooves overlaps with a lateral footprint of at least one of theone or more microvolumes; and performing a spectroscopic analysis withinthe overlapping lateral footprint. Optionally, the microfluidic devicecomprises a card shaped substrate.

In another embodiment, a method is provided for use with a microfluidicdevice, the method comprising: performing an experiment in a microvolumeof a microfluidic device; and performing a spectroscopic analysis usingan x-ray beam that traverses the microfluidic device such that materialwithin the microfluidic device that the x-ray beam traverses contains atleast as many electrons as is otherwise traversed when the x-ray beamtraverses the microfluidic device. Optionally, the material within themicrofluidic device that the x-ray beam traverses contains at leastthree, five, ten times or more times as many electrons as is otherwisetraversed when the x-ray beam traverses the microfluidic device.

In another embodiment, a method is provided for determiningcrystallization conditions for a material, the method comprising: takinga plurality of different crystallization samples in an enclosedmicrovolume, the plurality of crystallization samples comprising amaterial to be crystallized and crystallization conditions which varyamong the plurality of crystallization samples; allowing crystals of thematerial to form in the plurality of crystallization samples; andidentifying which of the plurality of crystallization samples comprise aprecipitate, oil or a crystal of the material. One or more dividers mayoptionally be positioned between different crystallization samples inenclosed microvolume to separate adjacent crystallization samples.

In another embodiment, a method is provided for determiningcrystallization conditions for a material, the method comprising: takinga plurality of different crystallization samples in a plurality ofenclosed microvolumes, each microvolume comprising one or morecrystallization samples, the crystallization samples comprising amaterial to be crystallized and crystallization conditions that varyamong the plurality of crystallization samples; allowing crystals of thematerial to form in plurality of crystallization samples; andidentifying which of the plurality of crystallization samples comprise aprecipitate, oil or a crystal of the material. One or more dividers mayoptionally be positioned between different crystallization samples inthe enclosed microvolumes to separate adjacent crystallization samples.

In another embodiment, a method is provided for determiningcrystallization conditions for a material, the method comprising: takinga microfluidic device comprising one or more lumens having microvolumedimensions and a plurality of different crystallization samples withinthe one or more lumens, the plurality of crystallization samplescomprising a material to be crystallized and crystallizationconditions-that vary among the plurality of crystallization samples;transporting the plurality of different crystallization samples withinthe lumens; and identifying a precipitate or crystal formed in the oneor more lumens. Transporting the plurality of different crystallizationsamples within the one or more lumens may be performed by a variety ofdifferent methods. For example, transporting may be performed by amethod selected from the group consisting of electrophoresis,electroosmotic flow and physical pumping. In one variation, transportingis performed by electrokinetic material transport.

In a variation according to this embodiment, at least one of the lumensoptionally comprises a plurality of different crystallization samples.One or more dividers may be positioned between different crystallizationsamples in at least one of the lumens to separate adjacentcrystallization samples.

Also according to this embodiment, the method may further compriseforming the plurality of different crystallization samples within theone or more lumens. The plurality of crystallization samples may becomprised in a single lumen or a plurality of lumens.

In another embodiment, a method is provided for determiningcrystallization conditions for a material, the method comprising: takinga microfluidic device comprising one or more lumens having microvolumedimensions and a plurality of different crystallization samples withinthe one or more lumens, the plurality of crystallization samplescomprising a material to be crystallized and crystallization conditionsthat vary among the plurality of crystallization samples; transportingthe plurality of different crystallization samples within the one ormore lumens; and identifying a precipitate or crystal formed in the oneor more lumens; and performing a spectroscopic analysis on theidentified precipitate or crystal while within the lumen.

The method may optionally further include forming the plurality ofdifferent crystallization samples within the one or more lumens. Theplurality of crystallization samples may be comprised in a single lumenor multiple lumens.

In another embodiment, a microfluidic method is provided comprising:delivering a first fluid to a first lumen of a microfluidic device and asecond, different fluid to a second lumen of the microfluidic device,the first and second lumens sharing a common wall that allows fordiffusion between the lumens over at least a portion of the length ofthe lumens; and having the first and second fluids diffuse between thefirst and second lumens.

In one variation according to this method, a composition of at least oneof the first and second fluids is varied so that the composition of atleast one of the first and second fluids varies along a length of thelumen.

In another variation according to this method, the composition of atleast one of the first and second fluids varies over time as it isdelivered to the lumen so that the fluid forms a gradient with regard toa concentration of at least one component of the fluid that changesalong a length of the lumen.

In another variation according to this method, the microfluidic devicecomprises a plurality of first and second lumens, the method comprisingdelivering first and second fluids to each of the plurality of first andsecond lumens.

In yet another variation according to this method, the same first andsecond fluids are delivered to each of the plurality of first and secondlumens.

In yet another variation according to this method, different first andsecond fluids are delivered to the plurality of first and second lumens.

It is noted that the first and second fluids may have a same ordifferent flow rate within the lumen. It is also noted that the firstand second fluids may each optionally comprise more than one differentfluid flow. The first and second fluids may also each optionallycomprise dividers that separate the fluid into a plurality of aliquotsseparated by the dividers.

In another variation according to this method, the method optionallyfurther comprises delivering a third fluid to a third lumen which sharesa common wall with at least one of the first and second lumens, thecommon wall allowing for diffusion between the third lumen and the firstor second lumen over at least a portion of the length of the lumens.

In another embodiment, a microfluidic device is provided that comprises:a substrate; a first lumen at least partially defined by the substrate;and a second lumen; wherein the first and second lumens share a commonwall with each other that allows for diffusion between the two lumensover at least a portion of the length of the two lumens. The common wallmay optionally comprise a membrane, gel, frit, or matrix that allows fordiffusion between the two lumens.

Also according to this embodiment, the device may further comprise athird lumen, the third lumen sharing a common wall with at least one ofthe first and second lumens so as to allow for diffusion between thelumens over at least a portion of the length of the lumens.

In another embodiment, a microfluidic device is provided that comprises:a substrate; a plurality of sets of lumens, each set comprising a firstlumen at least partially defined by the substrate, and a second lumen,wherein the first and second lumens share a common wall with each otherthat allows for diffusion between the two lumens over at least a portionof the length of the two lumens. The common wall may optionally comprisea membrane, gel, frit, or matrix that allows for diffusion between thetwo lumens.

According to this embodiment, the device may further comprise a thirdlumen, the third lumen sharing a common wall with at least one of thefirst and second lumens so as to allow for diffusion between the lumensover at least a portion of the length of the lumens.

Also according to this embodiment, the device may optionally comprise atleast 4, 8, 12, 24, 96, 200, 1000 or more sets of lumens.

A variety of different devices and methods are also provided that usecentrifugal force to cause fluid movement within a microfluidic device.

In one embodiment, a microfluidic method is provided that comprises:taking a microfluidic device comprising a plurality of microvolumes; andcausing movement of material in a same manner within the plurality ofmicrovolumes by applying centrifugal forces to the material.

In another embodiment, a microfluidic method is provided that comprises:taking a plurality of microfluidic devices, each device comprising aplurality of microvolumes; and causing movement of material in a samemanner within the plurality of microvolumes of the plurality of devicesby applying centrifugal forces to the material. Optionally, a samecentrifugal force is applied to each of the plurality of devices.

In a variation, the plurality of microfluidic devices may be stackedrelative to each other when the centrifugal forces are applied. Theplurality of microfluidic devices may also be positioned about arotational axis about which the plurality of microfluidic devices arerotated to apply the centrifugal forces.

In another embodiment, a microfluidic method is provided that comprises:taking a microfluidic device comprising a plurality of microvolumes; andphysically moving the device so as to effect a same movement of materialwithin the plurality of microvolumes. Physically moving the devicepreferably causes centrifugal force to be applied, for example, byrotation of the device about an axis.

According to this embodiment, the material moved in each of theplurality of microvolumes by movement of the device preferably has asame quantity.

In another embodiment, a microfluidic method is provided that comprises:taking a microfluidic device comprising a plurality of microvolumes; andaccelerating or decelerating a motion of the device so as to effect asame movement of material within the plurality of microvolumes.According to this embodiment, the motion of the device is optionally arotation of the device. In such instances, acceleration or decelerationmay be caused by a change in a rate of rotation of the device.

In another embodiment, a microfluidic device is provided that comprises:a substrate; and a plurality of microvolumes at least partially definedby the substrate, each microvolume comprising a first submicrovolume anda second submicrovolume that is in fluid communication with the firstsubmicrovolume when the device is rotated, the plurality of microvolumesbeing arranged in the device such that fluid in the firstsubmicrovolumes of multiple of the microvolumes are transported tosecond submicrovolumes of the associated microvolumes when the device isrotated.

According to this embodiment, the device may be designed so that atleast 4, 8, 12, 36, 96, 200, 1000 or more of the microvolumes aretransported to second submicrovolumes of the associated microvolumeswhen the device is rotated.

Also according to this embodiment, the device may be designed so thatthe volume of fluid delivered from the first submicrovolume to thesecond submicrovolume of a given microvolume upon rotation of the deviceis within 50%, 25%, 10%, 5%, 2%, 1% or less of the volume of fluiddelivered from the first submicrovolumes to the second submicrovolumesof any other microvolumes when a same volume of fluid is added to thefirst submicrovolumes.

In another embodiment, a microfluidic method is provided that comprises:taking a microfluidic device comprising a substrate, and a plurality ofmicrovolumes at least partially defined by the substrate, eachmicrovolume comprising a first submicrovolume and a secondsubmicrovolume where the first submicrovolume and second microvolume arein fluid communication with each other when the device is rotated;adding fluid to a plurality of the first submicrovolumes; and rotatingthe device to cause fluid from the plurality of first submicrovolumes tobe transferred to the second submicrovolumes in fluid communication withthe first submicrovolumes.

According to this embodiment, the device may be designed so that atleast 4, 8, 12, 24, 96, 200, 1000 or more of the microvolumes aretransported to second submicrovolumes of the associated microvolumeswhen the device is rotated.

Also according to this embodiment, the device may be designed so thatthe volume of fluid delivered from the first submicrovolume to thesecond submicrovolume of a given microvolume upon rotation of the deviceis within 50%, 25%, 10%, 5%, 2%, 1% or less of the volume of fluiddelivered from the first submicrovolumes to the second submicrovolumesof any other microvolumes when a same volume of fluid is added to thefirst submicrovolumes.

Also according to this embodiment, the method may be performed as partof performing an array crystallization trial. The array crystallizationtrial may involve the crystallization of a variety of differentmaterials including various biomolecules such as proteins.

In another embodiment, a microfluidic method is provided that comprises:taking a plurality of microfluidic devices, each comprising a substrate,and a plurality of microvolumes at least partially defined by thesubstrate, each sample microvolume comprising a first submicrovolume anda second submicrovolume where the first submicrovolume and secondsubmicrovolume are in fluid communication with each other when thedevice is rotated; adding fluid to a plurality of the firstsubmicrovolumes in the plurality of microfluidic devices; and rotatingthe plurality of microfluidic devices at the same time to cause fluidfrom the plurality of first submicrovolumes to be transferred to thesecond submicrovolumes in fluid communication with the firstsubmicrovolumes.

According to this embodiment, the plurality of microfluidic devices mayoptionally be stacked relative to each other during rotation. Theplurality of microfluidic devices may also be positioned about arotational axis about which the plurality of microfluidic devices arerotated. In one variation, the rotational axis about which the pluralityof microfluidic devices are rotated is positioned within the lateralfootprints of the plurality of microfluidic devices. In anothervariation, the rotational axis about which the plurality of microfluidicdevices are rotated is positioned outside of the lateral footprints ofthe plurality of microfluidic devices.

In yet another embodiment, a microfluidic device is provided thatcomprises: a substrate shaped so as to provide the device with an axisof rotation about which the device may be rotated; and a plurality ofmicrovolumes at least partially defined by the substrate, eachmicrovolume comprising a first submicrovolume and a secondsubmicrovolume that is in fluid communication with the firstsubmicrovolume when the device is rotated, the plurality of microvolumesbeing arranged in the device such that fluid in the firstsubmicrovolumes of multiple of the microvolumes are transported to thesecond submicrovolumes of the associated microvolumes when the device isrotated about the rotational axis. Optionally, the second microvolumesare lumens.

The device may optionally comprise a mechanism that facilitates thedevice being rotated about the rotational axis. For example, thesubstrate may define a groove or hole at the rotational axis thatfacilitates the device being rotated about the rotational axis.Optionally, a center of mass of the device is at the rotational axis andthe substrate defines a groove or hole at the rotational axis thatfacilitates the device being rotated about the rotational axis. In onevariation, the device is disc shaped, the substrate defining a groove orhole at the rotational axis of the disc that facilitates the devicebeing rotated about the rotational axis.

Also according to this embodiment, the method may be performed as partof performing an array crystallization trial. The array crystallizationtrial may involve the crystallization of a variety of differentmaterials including various biomolecules such as proteins.

In another embodiment, a microfluidic method is provided that comprises:taking a microfluidic device comprising a substrate, and a plurality ofmicrovolumes at least partially defined by the substrate, eachmicrovolume comprising a first and a second submicrovolume where thefirst and second submicrovolumes are in fluid communication with eachother when the device is rotated about a rotational axis of the device;adding fluid to a plurality of the first submicrovolumes; and rotatingthe device about the rotational axis of the device to cause fluid in thefirst submicrovolumes to be transferred to the second submicrovolumes.

Also according to this embodiment, the method may be performed as partof performing an array crystallization trial. The array crystallizationtrial may involve the crystallization of a variety of differentmaterials including various biomolecules such as proteins.

In another embodiment, a microfluidic device is provided that comprises:a substrate; one or more microvolumes at least partially defined by thesubstrate, each microvolume comprising a first submicrovolume, a secondsubmicrovolume where fluid in the first submicrovolume is transported tothe second submicrovolume when the device is rotated about a firstrotational axis, and a third submicrovolume where fluid in the firstsubmicrovolume is transported to the third submicrovolume when thedevice is rotated about a second, different rotational axis. The deviceitself include features to facilitate the rotation of the device aboutone or more rotational axes. The device may alternative be rotated aboutone or more rotational axes by the use of an external fixture.

In another embodiment, a microfluidic device comprising: a substrate;one or more microvolumes extending along a plane of the substrate, eachmicrovolume comprising a first submicrovolume, a second submicrovolumewhere fluid in the first submicrovolume is transported to the secondsubmicrovolume when the device is rotated about a first rotational axisthat is positioned further away from the second submicrovolume than thefirst submicrovolume, and a third submicrovolume where fluid in thefirst submicrovolume is transported to the third submicrovolume when thedevice is rotated about a second, different rotational axis that ispositioned further away from the third submicrovolume than the firstsubmicrovolume. Optionally, the substrate is card shaped. In suchinstances, the one or more microvolumes may optionally extend along asurface of the card shaped substrate.

In another embodiment, a microfluidic method is provided that comprises:taking a microfluidic device comprising a substrate and a plurality ofmicrovolumes at least partially defined by the substrate, eachmicrovolume comprising an first submicrovolume, a second submicrovolumewhere fluid in the first submicrovolume is transported to the secondsubmicrovolume when the device is rotated about a first rotational axis,and a third submicrovolume where fluid in the first submicrovolume istransported to the third submicrovolume when the device is rotated abouta second, different rotational axis; adding fluid to the firstsubmicrovolumes of the microvolumes; and in any order rotating thedevice about the first and second rotational axes to cause fluid fromthe first submicrovolumes to be transferred to the second and thirdsubmicrovolumes.

It is noted that the method may be performed as part of performing anarray crystallization trial. The array crystallization trial may involvethe crystallization of a variety of different materials includingvarious biomolecules such as proteins.

In another embodiment, a microfluidic device is provided that comprises:a substrate; and a plurality of microvolumes at least partially definedby the substrate, each microvolume comprising a first submicrovolume anda second submicrovolume in fluid communication with the firstsubmicrovolume when the device is rotated about a first rotational axis,wherein rotation of the device about the first rotational axis causes afixed volume to be transported to each of the second submicrovolumes.

According to this embodiment, the plurality of microvolumes mayoptionally further comprise one or more outlet submicrovolumes in fluidcommunication with the first submicrovolume.

Also according to this embodiment, the plurality of microvolumes mayoptionally further comprise one or more outlet submicrovolumes wherefluid in the first submicrovolume not transported to the secondsubmicrovolume when the device is rotated about a first rotational axisis transported to one or more one or more outlet submicrovolumes whenthe device is rotated about a second, different rotational axis.

In another embodiment, a microfluidic device is provided that comprises:a substrate; a first microvolume at least partially defined by thesubstrate comprising a first submicrovolume; a second submicrovolumewhere fluid in the first submicrovolume is transported to the secondsubmicrovolume when the device is rotated about a first rotational axis;and a second microvolume at least partially defined by the substratecomprising a third submicrovolume; a fourth submicrovolume where fluidin the third submicrovolume is transported to the fourth submicrovolumewhen the device is rotated about the first rotational axis; and whereinfluid in the second and fourth submicrovolumes are transported to afifth submicrovolume where the second and fourth submicrovolumes aremixed when the device is rotated about a second, different rotationalaxis.

According to this embodiment, the fifth submicrovolume may optionally bein fluid communication with the second and fourth submicrovolumes viathe first and third submicrovolumes respectively.

Also according to this embodiment, the device may further comprise oneor more outlet submicrovolumes in fluid communication with the first andthird submicrovolumes.

Also according to this embodiment, the device may further comprise oneor more outlet submicrovolumes in fluid communication with the first andsecond submicrovolumes where fluid in the first and thirdsubmicrovolumes not transported to the second and fourth submicrovolumeswhen the device is rotated about the first rotational axis istransported to one or more one or more outlet submicrovolumes when thedevice is rotated about a third, different rotational axis.

Also according to this embodiment, the device may further comprise atleast 4, 8, 12, 24, 96, 200, 1000, or more pairs of first and secondmicrovolumes.

Also according to this embodiment, the device may be designed such thatthe volume of fluid transported to any given second submicrovolume doesnot deviate from the volume of fluid transported to another secondsubmicrovolume by more than 50%, 25%, 10%, 5%, 2%, 1% or less.

The device may also optionally be designed so that any of the followingconditions are satisfied: the first rotational axis is positionedfurther away from the second and fourth submicrovolumes than the firstand third submicrovolumes; the first rotational axis about which themicrofluidic device is designed to be rotated is positioned within alateral footprint of the microfluidic device; and the first rotationalaxis about which the microfluidic device is designed to be rotated ispositioned outside of a lateral footprint of the microfluidic device.

In yet another embodiment, a microfluidic method is provided thatcomprises: taking a microfluidic device comprising a substrate, and aplurality of microvolumes at least partially defined by the substrate,each microvolume comprising a first submicrovolume and a secondsubmicrovolume in fluid communication with the first submicrovolume;adding fluids to the first submicrovolumes; and applying a centrifugalforce to the device to cause a same volume of fluid to be transported tothe second microvolumes from the first submicrovolumes.

Optionally, the microvolumes may further comprise an outletsubmicrovolume in fluid communication with the first submicrovolumes. Insuch instances, the method may further comprise transporting fluid inthe first submicrovolume to the outlet submicrovolume that was nottransported to the second submicrovolume when the centrifugal force wasapplied. The method may also further comprise transporting fluid in thefirst submicrovolume to the outlet submicrovolume that was nottransported to the second submicrovolume when the device is rotatedabout a first rotational axis by rotating the device about a second,different rotational axis.

Also according to the embodiment, the device may be designed such thatthe volume of fluid transported to any given second submicrovolume doesnot deviate from the volume of fluid transported to another secondsubmicrovolume by more than 50%, 25%, 10%, 5%, 2%, 1% or less.

In another embodiment, a microfluidic method is provided that comprises:taking a microfluidic device comprising a substrate, a first microvolumeat least partially defined by the substrate comprising a firstsubmicrovolume and a second submicrovolume where fluid in the firstsubmicrovolume is transported to the second submicrovolume when thedevice is rotated about a first rotational axis, and a secondmicrovolume at least partially defined by the substrate comprising athird submicrovolume and a fourth submicrovolume where fluid in thethird submicrovolume is transported to the fourth submicrovolume whenthe device is rotated about the first rotational axis, the microvolumesfurther comprising a fifth submicrovolume where fluid in the second andfourth submicrovolumes are mixed when the device is rotated about asecond, different rotational axis; adding a first fluid to the firstsubmicrovolume and a second fluid to the third submicrovolume; rotatingthe device about the first rotational axis to transport the first andsecond fluids to the second and fourth submicrovolumes; and rotating thedevice about the second rotational axis to transport the first andsecond fluids from the second and fourth submicrovolumes to the fifthsubmicrovolume.

In one variation, the fifth submicrovolume is in fluid communicationwith the second and fourth submicrovolumes via the first and thirdsubmicrovolumes respectively.

Optionally, the method further comprises removing fluid from the firstand third submicrovolumes that is not transported to the second andfourth submicrovolumes prior to rotating the device about the secondrotational axis.

Also according to the embodiment, the device may comprise a plurality ofpairs of first and second microvolumes and the volume of fluidtransported to any given second submicrovolume does not deviate from thevolume of fluid transported to another second submicrovolume by morethan 50%, 25%, 10%, 5%, 2%, 1% or less.

In another embodiment, a microfluidic method is provided that comprises:delivering first and second fluids to a lumen of a microfluidic devicesuch that the first and second fluids flow adjacent to each other withinthe lumen without mixing except for diffusion at an interface betweenthe first and second fluids, wherein the first fluid is different thanthe second fluid.

According to this embodiment, the composition of at least one of thefirst and second fluids is optionally varied over time as it isdelivered to the lumen so that the fluid forms a gradient with regard toa concentration of at least one component of the fluid that changesalong a length of the lumen.

According to this embodiment, the microfluidic device may comprise aplurality of lumens, the method optionally comprising delivering firstand second fluids to each of the plurality of lumens.

According to this embodiment, the same first and second fluids may bedelivered to each of the plurality of lumens. Alternatively, differentfirst and second fluids are delivered to the different lumens of theplurality of lumens. The first and second fluids may also have a same ordifferent flow rate within the lumen.

Also according to this embodiment, the first and second fluids may becombined to form different crystallization conditions for crystallizinga molecule such as a protein.

In another embodiment, a microfluidic method is provided that comprises:delivering first and second fluids to a lumen of a microfluidic devicesuch that the first and second fluids flow adjacent to each other withinthe lumen without mixing except for diffusion at an interface betweenthe first and second fluids, wherein the first fluid is different thanthe second fluid and a composition of at least one of the first andsecond fluids delivered to the lumen is varied so that the compositionof at least one of the first and second fluids within the lumen variesalong a length of the lumen.

In yet another embodiment, a microfluidic method is provided thatcomprises: delivering first, second and third fluids to a lumen of amicrofluidic device such that the first, second and third fluids flowadjacent to each other within the lumen without mixing except fordiffusion at an interface between the first, second and third fluids,wherein the first, second and third fluids are different than each otherand a composition of at least one of the first, second and third fluidsdelivered to the lumen is varied so that the composition of at least oneof the first, second, and third fluids within the lumen varies along alength of the lumen.

According to this embodiment, the composition of at least one of thefirst, second and third fluids may be varied over time as it isdelivered to the lumen so that the fluid forms a gradient with regard toa concentration of at least one component of the fluid that changesalong a length of the lumen.

Also according to this embodiment, the microfluidic device may comprisea plurality of lumens, the method comprising delivering first, secondand third fluids to each of the plurality of lumens.

The same or different first, second and third fluids may be delivered toeach of the plurality of lumens. Optionally, at least one of the first,second and third fluids have a different flow rate than another of thefluids within the lumen. Also, at least one of the first, second andthird fluids may have the same flow rate than another of the fluidswithin the lumen.

Also according to this embodiment, the first, second and third fluidsmay be combined to form different crystallization conditions forcrystallizing a molecule such as a protein. In one variation, the first,second and third fluids combine to form different crystallizationconditions, the second fluid comprising the material to be crystallizedand being positioned between the first and third fluids.

In regard to the various embodiments where a device is rotated about oneor more rotational axes, the device may optionally be designed so thatany or more of the following conditions are satisfied: the first andsecond rotational axes are laterally offset relative to each other; thefirst and second rotational axes are at an angle relative to each otherand intersect; the first and second rotational axes are at an anglerelative to each other and are laterally offset; the first and secondrotational axes are perpendicular to each other and intersect; the firstand second rotational axes are perpendicular to each other and arelaterally offset; the first and second rotational axes are at an angleof 45 degrees relative to each other and intersect; the first and secondrotational axes are at an angle of 45 degrees relative to each other andare laterally offset; and the first and second rotational axes areparallel and laterally offset relative to each other.

According to any of the embodiments employing centrifugal forces, thedevices may be designed so that material is optionally moved within atleast 4, 8, 12, 24, 96, 200, 1000 or more different microvolumes in asame manner when the centrifugal forces are applied.

Also according to any of the embodiments employing centrifugal forces,the devices may be designed so that the volume of fluid or othermaterial delivered to a submicrovolume in a given microvolume is within50%, 25%, 10%, 5%, 2%, 1% or less of the volume of fluid or othermaterial delivered to a corresponding submicrovolume in any othermicrovolume.

Optionally, the centrifugal forces are applied such that a samecentrifugal force is applied to material in each of the plurality ofmicrovolumes.

Optionally, the centrifugal forces are applied such that at least 0.01g, 0.1, 1 g, 10 g, 100 g or more force is applied to the material in thedevice to cause the material to move within the microvolumes.

Applying the centrifugal forces may be performed by rotating the device.Optionally, the centrifugal forces are applied by rotating the device atleast 10 rpm, 50 rpm, 100 rpm or more.

In regard to all of the above embodiments, unless otherwise specified,microvolumes may have a variety of shapes including, but not limited tolumens and microchambers. When a lumen is employed, the lumen optionallyhas a cross sectional diameter of less than 2.5 mm, optionally less than1 mm, and optionally less than 500 microns.

A variety of different substrates may be used to make the microfluidicdevices of the present invention. In one variation, the substratecomprises one or more members of the group consisting ofpolymethylmethacrylate, polycarbonate, polyethylene terepthalate,polystyrene, styrene copolymers, glass, and fused silica. In onevariation, the substrate is optically transparent.

According to each of the above embodiments, the experiment beingperformed may optionally be a crystallization of a molecule or material.The crystallization may optionally be of a biomolecule. Examples ofbiomolecules that may be crystallized include, but are not limited toviruses, proteins, peptides, nucleosides, nucleotides, ribonucleicacids, and deoxyribonucleic acids.

It is also noted that the material to be crystallized may contain one,two or more materials selected from the group consisting of viruses,proteins, peptides, nucleosides, nucleotides, ribonucleic acids,deoxyribonucleic acids, small molecules, drugs, putative drugs,inorganic compounds, metal salts, organometallic compounds and elements.In one variation, the material to be crystallized is a macromoleculewith a molecular weight of at least 500 Daltons.

In certain embodiments, a spectroscopic analysis is performed. Thespectroscopic analysis may optionally be-selected from the groupconsisting of Raman, UV/VIS, IR, x-ray spectroscopy, polarization, andfluorescent. In one particular variation, the spectroscopic analysis isx-ray spectroscopy. In a further particular variation, the x-rayspectroscopy is x-ray diffraction.

In some instances, the spectroscopic analysis involves an x-raytraversing the microfluidic device. In such instances, a groove may beemployed in the device that is sufficiently deep relative to the secondface of the substrate within the overlapping lateral footprint that whenthe portion of the microvolume within the overlapping lateral footprintcomprises a crystallization sample and an x-ray beam traverses the cardshaped substrate at the overlapping lateral footprint, the portion ofthe microvolume that the x-ray beam traverses contains at least half asmany electrons as is contained in the substrate where the x-ray beamtraverses. Optionally, the portion of the microvolume that the x-raybeam traverses contains at least as many electrons as is contained inthe substrate where the x-ray beam traverses. Preferably, the portion ofthe microvolume that the x-ray beam traverses contains at least three,five, ten times or more times as many electrons as is contained in thesubstrate where the x-ray beam traverses.

Each of the above embodiments may optionally include transportingmaterial within the microfluid device. Such transport may be performedby a variety of different methods. For example, transporting may beperformed by a method selected from the group consisting ofelectrophoresis, electroosmotic flow and physical pumping. In onevariation, transporting is performed by electrokinetic materialtransport. In some instances, transporting is performed by moving thedevice. This may be done by applying a centrifugal force, which in turnmay be performed by rotating the device about a rotational axis.

Each of the above embodiments may optionally include the use of one ormore dividers to separate aliquots of materials. In some instances, theseparated aliquots of materials correspond to separate experiments suchas crystallization trials. The dividers may be formed of a variety ofdifferent materials. For example, the dividers may be formed of apermeable, semi-permeable or impermeable material that may be a gas,liquid, gel, or solid. In one particular variation, the one or moredividers are selected from the group consisting of a membrane, gel,frit, and matrix.

The one or more dividers may form various interfaces including thoseselected from the group consisting of liquid/liquid, liquid/gasinterface, liquid/solid and liquid/sol-gel interface.

The one or more dividers optionally function to modulate diffusioncharacteristics between adjacent crystallization samples. For example,the one or more dividers may be formed of a semi-permeable material thatallows diffusion between adjacent crystallization samples.

These and other methods, devices, compositions and kits are describedherein.

BRIEF DESCRIPTION OF FIGURES

FIG. 1A illustrates a card shaped device housing microvolumes withopposing faces.

FIG. 1B illustrates an embodiment of a card shaped device where thethickness of the overall card is reduced adjacent a region where x-rayswill be incident in order to reduce the amount of material in the pathof the x-rays.

FIG. 1C illustrates a bottom up view of an embodiment of a card shapeddevice where grooves have been created so that less material is presentadjacent a region where x-rays will be incident.

FIG. 1D illustrates a cross sectional view of an embodiment of a cardshaped device where grooves have been created so that less material ispresent adjacent a region where x-rays will be incident.

FIG. 2 illustrates the generalized use of a microvolume dimensionedlumen to form crystallization samples and perform crystallization.

FIG. 3A illustrates various interconnections that may be formed betweendifferent lumens.

FIG. 3B illustrates how two sub-lumens extending from and joining with amain lumen may be used to effect mixing within the main lumen.

FIG. 3C illustrates the use of a dividing feature to separate a crystalcontaining crystallization experiment into two portions.

FIG. 3D illustrates different combinations of single and double portsthat may be combined for complex mixing, separation, diffusion andpurifications.

FIGS. 4A-4C provide several embodiments of performing crystallizationswithin a lumen.

FIG. 4A illustrates a crystallization mixture performed within a lumenpositioned between two dividers.

FIG. 4B illustrates a crystallization performed within a lumen wheremultiple crystallization conditions are simultaneously employed.

FIG. 4C illustrates a crystallization performed within a lumen where aseries of crystallization agents are set up for crystallization againsta series of substances to be crystallized.

FIG. 5A illustrates a crystallization performed within a lumen where oneor more of the elements of the crystallization experiment change along alength of the lumen. The change can occur discretely or continuously,and need not be changed in a simple linear method.

FIG. 5B illustrates a crystallization performed within a lumen where aseries of substances to be crystallized are in a single gradient.

FIG. 5C illustrates a crystallization performed within a lumen where aseries of crystallization agents can be assayed against a substance tobe crystallized.

FIG. 5D illustrates diffusion between various elements in acrystallization performed within a lumen.

FIG. 6A illustrates a crystallization performed within a lumen where asingle crystallization condition occupies an entire crystallizationspace.

FIG. 6B illustrates multiple crystallizations being performed within alumen where dividers are used between the crystallizations, the dividersbeing shown to have planar surfaces adjacent the crystallizations.

FIG. 6C illustrates multiple crystallizations being performed within alumen where dividers are used between the crystallizations, the dividersbeing shown to have curved, convex surfaces adjacent thecrystallizations.

FIG. 7A shows a device for performing a series of crystallizationswithin a series of lumens where each lumen comprises a loading andunloading port and a lumen body interconnecting the ports.

FIG. 7B shows a cross section of a device for performing acrystallization within a lumen where the lumen is not enclosed.

FIG. 7C shows a cross section of a device for performing acrystallization within a lumen where the lumens are rectangular inshape.

FIG. 7D shows a cross section of a device for performing acrystallization within a lumen where the lumens are curved or tubular inshape.

FIG. 7E shows a device for performing crystallizations within a seriesof lumens where the lumens are loaded with samples that are separated bydivider or modifier segments. It should be appreciated that eachdiscrete sample may have conditions that are potentially unique andunrelated to adjacent samples. The dividers or modifiers positionedbetween the samples can be permeable, semi-permeable or impermeable.

FIG. 8A shows a device for performing a series of differentcrystallizations within a series of lumens where each lumen comprises aloading and unloading port and a lumen body interconnecting the ports.

FIG. 8B illustrates a single lumen in which a barrier is adjacent to thecrystallization condition bounded by second barrier.

FIG. 8C illustrates a lumen comprising a more complex design ofcrystallizations than the lumen shown in FIG. 8B.

FIG. 8D illustrates a multi-component crystallization being performed ina single lumen.

FIG. 9A shows an embodiment of a device for performing a series ofdifferent crystallizations within a series of lumens where each lumencomprises a loading and unloading port and a lumen body interconnectingthe ports.

FIG. 9B illustrates an enlargement of a lumen of the device shown inFIG. 9A which illustrates some of the different simultaneous diffusionsthat are made possible by the invention.

FIG. 9C illustrates the device shown in FIG. 9B where diffusion occurredthrough the barrier to form a gradient from condition to condition.

FIG. 9D illustrates crystals forming at different locations after thediffusion shown in FIG. 9C.

FIG. 10A illustrates an embodiment of a device comprising first andsecond lumens where the first and second lumens share a common wall thatallows for diffusion between the lumens over at least a portion of thelength of the lumens.

FIG. 10B illustrates an embodiment of a device comprising first, secondand third lumens where the first, second and third lumens share a commonwall that allows for diffusion between the lumens over at least aportion of the length of the lumens.

FIG. 10C illustrates a crystallization experiment loaded into a doublelumen device such as the device shown in FIG. 10A.

FIG. 10D illustrates diffusion having occurred both through thesemi-permeable internal divider, as well as through the permeable orsemi-permeable wall of the device shown in FIG. 10C.

FIG. 10E illustrates the experiment shown in FIG. 10D where a series ofcrystal growths have occurred after some diffusion has occurred.

FIG. 10F illustrates a device with multiple lumens that may each beseparated by permeable or semi-permeable wall.

FIG. 11A illustrates a single lumen with integral mixing and harvestingchannels.

FIG. 11B shows an embodiment of a device for performing a series ofdifferent crystallizations within a series of lumens where each lumencomprises integral mixing and harvesting channels.

FIG. 12A illustrates a device comprising a series of lumens, each lumenhaving attached to it an array of individual crystallization cells, eachcell having at least one separate inlet or outlet and at least onechannel connecting the cell to the lumen.

FIG. 12B illustrates an embodiment of an individual crystallization cellshown in FIG. 12A.

FIG. 12C illustrates an embodiment of an individual crystallization cellshown in FIG. 12A where the cell comprises a crystallization agent and asubstance to be crystallized.

FIG. 13 illustrates a device for forming crystallizations by rotation ofthe device.

FIG. 14 illustrates a device that is designed to move fluids within thedevice by centrifugal force.

FIGS. 15A-15G illustrate an embodiment of a centrifugally drivencrystallization device.

FIG. 15A illustrates a repeating unit of the centrifugal array.

FIG. 15B illustrates a process for using a centrifugal device.

FIG. 15C illustrates the effect of centrifugal force on the samples thatare loaded in the centrifugal device illustrated in FIG. 15B.

FIG. 15D illustrates what happens when the centrifugal force vector ischanged such that the force now directs the excess crystallization agentand excess material to be crystallized toward the waste ports via therespective waste channels.

FIG. 15E illustrates each channel of the centrifugal device filled topoint V, resulting in precise volume measurements.

FIG. 15F illustrates what happens when the centrifugal force vector hasbeen altered to align in the direction shown.

FIG. 15G illustrates crystallization chamber filled with the combinationof the material to be crystallized and the crystallization agent, oragents.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to various methods, devices and kitsrelating to microfluidics.

One particular aspect of the present invention relates to the use ofthese methods and devices for forming crystallization samples,transporting crystallization samples, and crystallizing materialstherein, particularly on a microvolume scale, high throughput manner.Distinguishing the present invention in this regard is the performanceof the crystallizations in very small, substantially enclosed volumesformed by or within a substrate, referred to herein as an “enclosedmicrovolume”. Other aspects of the present invention will be understoodby one of ordinary skill in view of the teachings provided herein.

It is noted that many of the particular embodiments are described hereinin regard to performing crystallization experiments. However, it shouldbe understood that many of the operations involved in performingcrystallization experiments (e.g., measuring, mixing, fluid flow andanalysis) made possible by the various devices and methods of thepresent invention have applications outside of performingcrystallization experiments and should therefore not be limited tocrystallization experiments.

A “crystallization sample”, as the term is used herein, refers to amixture comprising a material to be crystallized. The crystallizationincludes such other components in the mixture to cause or at leastattempt to cause crystals of the material to be formed in the mixture.

According to the present invention, crystallization samples are formed,transported, and crystallization attempts conducted in enclosedmicrovolumes. These enclosed microvolumes comprise one or more lumensand optionally microchambers in fluid communication with the lumens. Thelumens are enclosed within a substrate. When employed, microchambers areenclosed microvolumes defined within the substrate in fluidcommunication with the lumens. The lumens and microchambers provide anencased environment within which crystallization samples may be formed,and crystallization attempts performed and analyzed.

The term “lumen” as the term is used herein, refers to any elongated,enclosed volume formed at least partially by a substrate. The lumenpreferably has a cross sectional diameter of less than 2.5 mm,preferably less than 1 mm and more preferably less than 500 microns. Inone variation, the lumen has a cross sectional diameter between 0.1microns and 2.5 mm, preferably between 0.1 microns and 1 mm, andpreferably between 0.1 and 500 microns. Aside from openings in thelumen, most typically adjacent the proximal and distal ends of thelumen, the lumen provides an enclosed environment in which to form,transport, conduct, and optionally analyze crystallizations.

Mass flow may be reduced by controlling the length of thecrystallization volume within the microlumens. This serves to reduce theforces driving convection currents within the crystallization condition.By minimizing the length of the crystallization volume within themicrolumens, facile control of the degree of convection currents withinthe microlumen is controlled.

In certain instances, it may be desirable for the lumen to be in fluidcommunication with one or more microchambers. A “microchamber”, as theterm is used herein, refers to a volume in fluid communication with alumen that has a larger cross sectional area than the lumen.

By forming crystallization conditions and performing crystallizationswithin the small, relatively sealed volumes defined by the enclosedmicrovolumes of the lumens and microchambers, a variety of differentadvantages are provided.

One advantage provided by conducting crystallizations in enclosedmicrovolumes is that it facilitates parallel screening of many materialsat once or a material in many conditions at once, or a combinationthereof.

A further advantage provided by the small volumes associated withperforming crystallizations in enclosed microvolumes is that it enablesthe conservation of the material to be crystallized, thereby enablinggreater numbers of crystallization conditions to be sampled using agiven amount of material. By achieving higher densities ofcrystallization conditions, advancements in crystal analysis areobtained.

A further advantage provided by performing crystallizations according tothe present invention is a reduction in evaporation during thepreparation and performance of the crystallization. As a result,crystallization conditions can be more precisely controlled and remainstable for longer periods of time. Crystallizations can also beconducted over a wider range of temperature conditions since losses dueto evaporation are significantly curtailed.

A further advantage provided by performing crystallizations according tothe present invention is a further reduction in the space requirementsfor performing crystallizations. More specifically, the presentinvention allows multiple crystallizations to be performed in a denserformat. This allows the device within which the crystallizations areperformed to be smaller and allows more crystallizations to be performedin a single device. For example, when in situ crystallizations areperformed in a thin cassette or card, the crystallizations may bedensely packed, allowing for rapid and efficient analysis of thecrystallization conditions.

A further advantage provided by performing crystallizations according tothe present invention is the more rapid equilibration times that may beachieved by further reducing crystallization volumes.

A further advantage provided by performing crystallizations according tothe present invention is the number of parallel experiments that may beperformed. For example, embodiments of the present invention provide forthe use of at least 4, 8, 12, 24, 96, 200, 1000 or more differentmicrovolumes in parallel. In regard to the use of centrifugal forces,the devices may be designed so that material is moved within at least 4,8, 12, 24, 96, 200, 1000 or more different microvolumes in a same mannerwhen the centrifugal forces are applied.

Yet a further advantage provided by performing crystallizationsaccording to the present invention is the precision with which fluid andmaterial can be transported. For example, certain embodiments usecentrifugal forces to transport materials. By being able to closelycontrol the sizes of the microvolumes, devices can be designed so thatthe volume of fluid delivered to a given submicrovolume of a givenmicrovolume upon rotation of the device is within 50%, 25%, 10%, 5%, 2%,1% or less of the volume of fluid delivered to submicrovolumes of othermicrovolumes.

As will be evident from the foregoing description of the operation ofthe devices of the present invention, a further advantage provided issimplified material handling.

1. Materials To Be Crystallized

While problems associated with crystal growth addressed by the presentinvention are of particular interest for proteins and otherbiomolecules, it is a general problem of all crystal forming materials.The materials to be crystallized may be any substance capable ofcrystallizing or co-crystallizing. For example, the material to becrystallized may contain one, two or more materials selected from thegroup consisting of viruses, proteins, peptides, nucleosides,nucleotides, ribonucleic acids, deoxyribonucleic acids, ligands, smallmolecules, drugs, putative drugs, inorganic compounds, metal salts,organometallic compounds and elements and mixtures and combinationsthereof.

The materials to be crystallized may be any material for which a crystalstructure is needed. Determining high-resolution structures of materialsby a high-throughput method such as the one of the present invention canbe used to accelerate the analysis of materials, especially drugdevelopment.

The material to be crystallized may also be a molecule for which acrystalline form of the molecule is needed. For example, it may bedesirable to create a crystalline form of a molecule or to identify newcrystalline forms of a molecule. In some instances, particularcrystalline forms of a molecule may have greater biological activity,dissolve faster, decompose less readily, and/or be easier to purify.

The material to be crystallized may also be a-combination of substancesfor the production of co-crystals. The co-crystals can comprise any twoof a small molecule, a drug, a ligand, a substrate, an inhibitor, aguest chemical, protein, nucleotide, or a protomer. The substances canbe a plurality of small molecules, drugs, ligands, substrates,inhibitors, guest chemicals, proteins, or a protomers.

The material to be crystallized is preferably a macromolecule such as aprotein but may also be other types of macromolecules. The moleculepreferably has a molecular weight of at least 500 Daltons, morepreferably at least 1000 Daltons, although smaller molecular weightmolecules may also be crystallized.

2. Construction of Enclosed Microvolumes

The construction, design and operation of various different microfluidicdevices have been described in literature and are thus known in the art.For example, U.S. Pat. Nos. 5,126,022; 5,296,114; 5,180,480; 5,132,012;and 4,908,112 are examples of references detailing the design andconstruction of lumens and microchambers in a substrate. Other examplesof references include Harrison et al., “Micromachining a MiniaturizedCapillary Electrophoresis-Based Chemical Analysis System on a Chip,”Science (1992) 261: 895; Jacobsen et al., “Precolumn Reactions withElectrophoretic Analysis Integrated on a Microchip,” Anal. Chem. (1994)66: 2949; Effenhauser et al., “High-Speed Separation of AntisenseOligonucleotides on a Micromachined Capillary Electrophoresis Device,”Anal. Chem. (1994) 66:2949; and Woolley & Mathies, “Ultra-High-Speed DNAFragment Separations Using Capillary Array Electrophoresis Chips,”P.N.A.S. USA (1994) 91:11348. Further examples of different microfluidicdevices include, but are not limited to those described in: U.S. Pat.Nos. 6,306,273, 6,284,113, 6,176,962, 6,103,537, 6,093,296, 6,074,827,6,007,690, 5,858,188, 5,126,022, 5,750,015, 5,935,401, 5,770,029assigned to Aclara, Inc.; U.S. Pat. Nos. 6,321,791, 6,316,781,6,316,201, 6,306,272, 6,274,337, 6,274,089, 6,267,858, 6,251,343,6,238,538, 6,235,175, 6,221,226, and 6,186,660 assigned to Caliper,Inc.; PCT Application Nos. WO 01/94635, WO 01/75176, WO 01/67369, WO01/32930, WO 01/01025, WO 99/61888, each assigned to Fluidigm, Inc.;U.S. Pat. Nos. 6,319,472, 6,238,624 assigned to Nanogen, Inc.; U.S. Pat.Nos. 6,290,685 assigned to 3M Corp.; as well as U.S. Pat. Nos.6,261,430, 6,251,247, 6,236,945, 6,210,986, 6,176,990, 6,007,690,6,074,827, 6,056,860, 6,054,034, 5,885,470, 5,858,195, 5,750,015,5,571,410, 5,580,523, 5,296,114, 5,180,480, 5,132,012, 5,126,022,4,891,120, and 4,908,112.

It should be understood that these numerous examples are only intendedto be illustrative in regard how enclosed microvolumes according to thepresent invention may be constructed, designed and operated inconjunction with the present invention.

Transport of material within the microfluidic devices of the presentinvention may be performed by any mode of transport available tomicrofluidic devices including, but not limited to electrophoresis,electroosmotic flow and physical pumping. In one variation, transportingis performed by electrokinetic material transport. A novel feature ofcertain embodiments of the present invention, discussed herein ingreater detail, is the use of centrifugal force to transport material,for example by rotating the device about a rotational axis.

The enclosed microvolumes may be formed in any substrate within whichmicrovolumes may be formed. Examples of suitable substrates include, butare not limited to glass, fused silica, acrylics, thermoplastics, andthe like. The various components of the integrated device may befabricated from the same or different materials, depending on theparticular use of the device, the economic concerns, solventcompatibility, optical clarity, color, mechanical strength, and thelike.

For applications where it is desired to have a disposable device, due toease of manufacture and cost of materials, the device will typically befabricated from a plastic. For ease of detection and fabrication, theentire device may be fabricated from a plastic material that isoptically transparent, as that term is defined above. Particularplastics finding use include polymethylmethacrylate, polycarbonate,polyethylene terepthalate, polystyrene or styrene copolymers, and thelike. It is noted that these various materials may be used alone or incombination to form the devices of the present invention.

The substrate comprising the enclosed microvolumes may be in any form,e.g., a tube, a card, a chip or a block. The substrate is preferably inthe form of a card. The card preferably has a face sized less than 12cm×8.5 cm.

The enclosed microvolumes may be formed by any process by which anenclosed lumen or chamber may be created in a material. For example, theshape of the substrate and the enclosed microvolumes may be formed bythermoplastic injection molding, micromolding, punching, milling, anysolid free form technology, such as three dimensional printing, or othertypes of manufacturing technologies for plastics, such as micromolding,embossing, laser drilling, extrusion, injection, or electron depositionmachining, glass or silicon, conventional silicon processing technology,such as photolithography, deep reactive ion or wet etching, electronbeam machining, micromachining, electro-discharge machining, reactioninjection molding.

It is noted that the substrate comprising the enclosed microvolume maybe formed of a single material, such as a block or a card.Alternatively, one or more materials may be brought together to form theenclosed microvolume. This typically involves having a portion of themicrovolume be formed by a first substrate (e.g., photolithography on asurface of the first substrate). A second substrate is brought togetherwith the first substrate to complete the definition of the enclosedmicrovolume. The act of combining the first and second substrates cancause the material to be crystallized to be enclosed. The act ofcombining can also cause mixing to occur. The substrate is preferablyoptically clear, transparent, translucent or opaque.

The substrate is preferably formed of a material that allows for variousspectroscopic analyses (e.g., Raman, UV/VIS, IR or x-ray spectroscopy,polarization, fluorescent, and with suitable designs, x-ray diffraction)to be performed in situ. In one particular variation, the spectroscopicanalysis is x-ray spectroscopy. In a further particular variation, thex-ray spectroscopy is x-ray diffraction.

In order to improve the performance of the device for performing in situx-ray spectroscopy such as x-ray diffraction and other forms ofspectroscopy where an x-ray is caused to traverse the substrate, thenumber of electrons in the path of the x-ray beam of the material beinganalyzed should be maximized relative to the number of electrons that isotherwise in the path of the x-ray beam.

The number of electrons of the device that are traversed can be reducedby choosing materials to form the device that have a low atomic number(Z), or a low density. Examples of materials that are preferably usedfor reducing the number of electrons in the substrate material includelow density plastics such as polystyrene, polyethylene, polypropyleneand other carbon based polymers. Silicon materials, such as siliconwafers, glass, including borosilicate and soda glass, and aerogels canbe suitable materials. Optically opaque materials that are suitableinclude Beryllium, plastic films and plastics.

A key parameter R, corresponds to a ratio between the number ofelectrons within the sample, (e.g., the precipitate, oil, crystal andoptionally other contents of the microvolume) that the x-ray traverses,and the sum of the electrons contained in the support material and thelid, or sealing material that the x-ray traverses.${R = \frac{\sum\quad\left\lbrack e^{-} \right\rbrack_{Crystal}}{\sum\left\lbrack e^{-} \right\rbrack_{Cassette}}},$

-   -   where the number of electrons in the x-ray beam, [e⁻], is        calculated by multiplying the density of the material in grams,        by thickness of the material and the area of the x-ray beam at        the microlumen, which gives the mass in grams, X, of the        microlumen material in the x-ray beam. This can be converted        into the number of electrons by multiplying the mass in grams by        Avogadro's number, N, and dividing by the molecular weight of        the material, MW. i.e., [e⁻]=X*N/MW.

The contents of the microvolume that the x-ray beam traverses preferablycontains at least half as many electrons as is contained in thesubstrate where the x-ray beam traverses. More preferably, the portionof the microvolume that the x-ray beam traverses contains at least one,three, five, ten times or more as many electrons as is contained in thesubstrate where the x-ray beam traverses.

In some instances, it is a particular precipitate, oil, or crystal thatis being analyzed. It is also preferred that the particular precipitate,oil, or crystal that the x-ray beam traverses contains at least half asmany electrons as is contained in the device where the x-ray beamtraverses. More preferably, the particular precipitate, oil, or crystalthat the x-ray beam traverses should contain at least one, three, fiveten times or more as many electrons as is contained in the device wherethe x-ray beam traverses.

The number of electrons in the path of the incident x-rays can bereduced by minimizing the mass of material in the path. Accordingly, thesubstrate enclosing the microvolumes preferably contains as littlematerial as possible in the direction of the path of the x-rays. Asillustrated in FIG. 1A, the device housing the microvolumes 100 willmost commonly have a card shape 102 with opposing faces 104 and 106.Walls 108, 110 (shown in FIGS. 1B and 1C) adjacent the opposing facesdefine a portion of the microvolume. X-rays 112 will typically traversethe card substantially perpendicular to the opposing faces in order tominimize the path length across the card, that path length being definedlargely by the thickness of walls 108, 110. It is desirable for the cardto have sufficient thickness so that it will be sufficiently rigid fornecessary handling. However, by reducing the amount of material formingthe walls 108, 110 adjacent a portion of the microvolume where x-rayswill be incident, one can reduce the amount of mass in the path of thex-rays.

FIG. 1B illustrates an embodiment where the thickness of the overallcard is reduced adjacent a region where x-rays will be incident in orderto reduce the amount of material in the path of the x-rays.

FIGS. 1C and 1D illustrate an embodiment where less material is presentadjacent a region where x-rays will be incident in order to reduce theamount of material in the path of the x-rays. This may be accomplishedby forming a card as shown in FIG. 1C with grooves 114 on one or bothsides adjacent where x-rays will be incident. As used herein, a grooverefers to any recess formed in the substrate so that the thickness ofthe device is reduced.

If the microvolume is closely adjacent one face of the card, a groovemay be formed adjacent the opposite side of the card as shown in thecross sectional view provided by FIG. 1D. It is noted that the card maybe formed with the groove or may be formed without the groove and thenmaterial may be removed from the card to create the groove.

At least a portion of a groove is preferably positioned within a lateralfootprint of the microvolume where it is desired to have the x-ray beamtraverse the device. Accordingly, one embodiment of the inventionrelates to a microfluidic device that comprises: a card shaped substratehaving first and second opposing faces; one or more microvolumes atleast partially defined by a first face of the card shaped substrate;and one or more grooves at least partially defined by a second face ofthe card shaped substrate; wherein a lateral footprint of at least aportion of the one or more grooves overlaps with a lateral footprint ofat least one of the one or more microvolumes.

At the overlap, the groove is preferably sufficiently deep that an x-raybeam traversing the device encounters at least half as many electronswithin the microvolume as the remainder of the device that the x-raybeam traverses. More preferably, the x-ray beam traversing the deviceencounters at least one, three, five, ten times or more as manyelectrons within the microvolume as the remainder of the device that thex-ray beam traverses.

As illustrated in FIG. 1C, the microvolume may be a lumen. The groovemay have a longitudinal axis that is aligned with a longitudinal axis ofthe lumen adjacent the overlapping lateral footprint (shown in FIG. 1C).This provides a greater area for the x-ray beam to traverse within theoverlap. It is recognized, however, that the groove may not have alongitudinal axis or may have a longitudinal axis that is misaligned,optionally to the extent of being perpendicular to a longitudinal axisof the lumen adjacent the overlapping lateral footprint.

By reducing the amount of substrate encountered by an x-ray beam, forexample by using devices with grooves such as those shown in FIGS. 1Cand 1D, methods may be performed according to the present inventioncomprising: performing an experiment in a microvolume of a microfluidicdevice; and performing a spectroscopic analysis using an x-ray beam thattraverses the microfluidic device such that material within themicrofluidic device that the x-ray beam traverses contains at least asmany electrons as is otherwise traversed when the x-ray beam traversesthe microfluidic device. Optionally, the material within themicrofluidic device that the x-ray beam traverses contains at leastthree, five, ten times or more as many electrons as is otherwisetraversed when the x-ray beam traverses the microfluidic device.

As will be illustrated herein, crystallization conditions may be formedby delivering different components to a single lumen or by deliveringdifferent components to a given lumen or microchamber from multipledifferent lumens. In this regard, the multiple different lumens arepreferably interconnected.

The cross sectional shape of the lumen may stay the same or may varyalong the length of the lumen. Optionally, the lumen may be connected toone or more chambers to which material from the lumen is delivered orfrom which material is delivered to the lumen. It is noted thatcrystallizations may also be performed in the chambers after material isdelivered via the lumen to the chamber.

The lumen may have a variety of cross sectional geometries. For example,the cross-sectional geometry of the lumen may be circular,semi-circular, ovoid, “U” shaped, square, or rectangular, or one or morecombinations thereof. Preferably, the cross sectional area of the lumenis small relative to the length of the lumen. This serves to reduceconvection currents within liquids passing within the lumen. Convectioncurrents may be further reduced by the use of thixotropic agents, suchas silica gel, agarose, other polysaccharides and polymers.

3. Layout and Use of Microsized Lumens for Performing CrystallizationTrials

Various methods and devices are provided for performing crystallizationtrials in microfluidic devices. For example, in one embodiment, a methodis provided for determining crystallization conditions for a material,the method comprising: taking a plurality of different crystallizationsamples in an enclosed microvolume, the plurality of crystallizationsamples comprising a material to be crystallized and crystallizationconditions that vary among the plurality of crystallization samples;allowing crystals of the material to form in the plurality ofcrystallization samples; and identifying which of the plurality ofcrystallization samples comprise a precipitate, oil or a crystal of thematerial.

In another embodiment, a method is provided for determiningcrystallization conditions for a material, the method comprising: takinga plurality of different crystallization samples in a plurality ofenclosed microvolumes, each microvolume comprising one or morecrystallization samples, the crystallization samples comprising amaterial to be crystallized and crystallization conditions which varyamong the plurality of crystallization samples; allowing crystals of thematerial to form in plurality of crystallization samples; andidentifying which of the plurality of crystallization samples comprise aprecipitate, oil or a crystal of the material.

In another embodiment, a method is provided for determiningcrystallization conditions for a material, the method comprising: takinga microfluidic device comprising one or more lumens having microvolumedimensions and a plurality of different crystallization samples withinthe one or more lumens, the plurality of crystallization samplescomprising a material to be crystallized and crystallization conditionsthat vary among the plurality of crystallization samples; transportingthe plurality of different crystallization samples within the lumens;and identifying a precipitate or crystal formed in the one or morelumens. Transporting the plurality of different crystallization sampleswithin the one or more lumens may be performed by a variety of differentmethods. For example, transporting may be performed by a method selectedfrom the group consisting of electrophoresis, electroosmotic flow andphysical pumping. In one variation, transporting is performed byelectrokinetic material transport. In a variation according to thisembodiment, at least one of the lumens optionally comprises a pluralityof different crystallization samples.

In another embodiment, a method is provided for determiningcrystallization conditions for a material, the method comprising: takinga microfluidic device comprising one or more lumens having microvolumedimensions and a plurality of different crystallization samples withinthe one or more lumens, the plurality of crystallization samplescomprising a material to be crystallized and crystallization conditionsthat vary among the plurality of crystallization samples; transportingthe plurality of different crystallization samples within the one ormore lumens; and identifying a precipitate or crystal formed in the oneor more lumens; and performing a spectroscopic analysis on theidentified precipitate or crystal while within the lumen.

The method may optionally further include forming the plurality ofdifferent crystallization samples within the one or more lumens. Theplurality of crystallization samples may be comprised in a single lumenor multiple lumens.

According to any of these embodiments, the methods may further compriseforming the plurality of different crystallization samples within theone or more lumens. The plurality of crystallization samples may becomprised in a single lumen or a plurality of lumens.

Also according to any of these embodiments, one or more dividers mayoptionally be positioned between different crystallization samples inthe enclosed microvolumes to separate adjacent crystallization samples.

A generalized use of a microfluidic sized lumen to form crystallizationsamples and perform crystallization attempts is illustrated in regard toFIG. 2. As shown in step A of FIG. 2, an enclosed lumen 201 is providedsuch that the lumen 201 has at least one opening 202A adjacent a firstend of the lumen and at least one opening 202B adjacent a second end ofthe lumen. A crystallization experiment 203 is introduced into the lumen201 via one of the openings, as shown in step B. This material may be apre-formed crystallization experiment, consisting of a material to becrystallized and one or more crystallization agents, or it may be amaterial to be crystallized that will undergo a diffusion experiment,wherein material will be transferred either through vapor or liquiddiffusion. Step C of the figure shows the crystallization experimentproceeding such that a portion of the material either crystallizes intoa crystal 204 or a plurality of crystals, microcrystals, needles,precipitates or other solids, or the material remains in solution.

If a crystal forms, as shown in step D of the figure (shown as 205),then the crystal, precipitate, oil, etc. may be examined in situ, forexample, as shown in steps E-H. Examination may be performed by anyavailable method, including, but not limited to spectroscopically,visually, or if the crystallization channel is suitably designed, bydirect exposure of x-rays. As shown by the arrows leading from step D,the crystal or crystallization mixture may be harvested from the lumen.

Steps E-H show different processing steps that may be performed on thecrystal or crystallization mixture. Step E illustrates a crystal beingexamined within a lumen via x-ray diffraction by using an x-ray source208 suitable for diffraction experiments, which is suitably focused andcollimated to pass through the material to be examined. The diffractedx-rays can then be examined through the use of a suitable x-ray detector206, which can be x-ray film, one dimensional x-ray detectors, twodimensional (area) detectors, or an electronic x-ray detector orscintillator. Alternatively, as shown in step F, the crystal 205 can bemanipulated within the crystallization channel. This enables theharvesting of the crystal as shown in step G, wherein the crystalcontaining crystal experiment is brought to an outlet of the crystalchannel.

As shown, the crystal can be harvested into an intermediate device, ormay be harvested directly with a mounting suitable for x-raydiffraction. This mounting can be a loop 209, as shown in step G, or itcan be a capillary suitable for x-ray mounting, or a fiber, or aspatula. These techniques for harvesting and manipulating crystals arewidely known. Once the crystal is harvested, the crystal can then betransported to an x-ray diffraction experiment shown as step H where thecrystal can be mounted in a position to facilitate the diffractionexperiment. It should be appreciated that the material to be analyzedmay not be a single crystal. For example, the material may be twinnedcrystals, or a plurality of crystals grouped together, or a number ofloose crystals, a precipitate, or an oil that can then be examined forcrystalline elements.

The crystallization drop 203 can be created within the microlumen, or itcan be mixed outside of the channel and introduced into the channel. Theactual method for loading the channels will vary depending upon thenecessities of the experiment. A crystallization mixture can be formedby the use of a syringe, such as a Hamilton syringe, or via a parallelrobotic system such as the Tecam, wherein the relevant volume ofmaterial to be crystallized is drawn up into the syringe and then therelevant volume of the crystallization agent can be drawn up. Thematerial may be dispensed directly into the loading port 202, or may bedispensed into or onto an intermediate surface or container for mixing.The material can be applied to the inlet port under pressure from thesyringe, or may be loaded onto the upper surface of 201, such that thedroplet covers the inlet port 202. The droplet can then be loaded intothe microlumen by the application of a pressure difference to 202 and202′, either through pressure at 202 or through vacuum at 202′.Similarly, after the crystal has grown, the application of a pressuredifference, either directly, or indirectly through a pressure transferfluid, such as mineral oil or buffer, the crystal can be moved to theoutlet port 202′, for harvesting as shown above in steps 1-3 of FIGS. 2Gand 2H.

It is noted that a given lumen may have multiple lumens interconnectingwith it or extending from it. For example, as shown in FIG. 3A, a lumen301 may have two inlet ports 302A and 302B and a junction 303 that mayform an acute, perpendicular or obtuse intersection. A perpendicularintersection is illustrated in FIG. 3A where the intersection 304 ofchannels 302C and 302D is formed perpendicularly.

FIG. 3B illustrates how two sub-lumens extending from and joining with amain lumen may be used to effect mixing within the main lumen. Material305A in one sub-lumen of the main lumen and material 305B in the secondsub-lumen extending from the main lumen are joined and mixed togetherinto a single volume 306 by the geometry of the interconnectingchannels. Depending upon the particular application, the lumens andsub-lumens can be designed to affect differing levels of mixing and thealteration of the interface between the two substances. Obviously, thelumens may possess both combining features or dividing features or acombination thereof, or depending on the absolute and relative flowscombining features that function as dividing features under alteredfluid flows.

FIG. 3C illustrates the use of a dividing feature 303 to separate acrystal containing crystallization experiment 307 into two portions 308Aand 308B. It should be understood that the relative volumes in 308A and308B may be readily attained by, suitable design or practice, byachieving differential fluid flows.

It should also be appreciated that different combinations of single anddouble ports may be combined for complex mixing, separation, diffusionand purifications as illustrated in FIG. 3D. For example, as shown,ports 309A and 309B meet at junction 310A. Similarly, ports 309C, 309Dmeet at junction 310B. The sub-lumens from junctions 310B and 310C canintersect at 310A.

To generate the result shown in FIG. 3A, one might apply a sample toinlet port 302, block port 302′, and apply a positive pressuredifference between port 302 and the pressure within 301. This can beaffected by applying a small vacuum at 301, by the removal of materialfrom 301 hydraulically, or by the application of pressure at 302, or bythe application of centrifugal force with a component along 301. Thedroplet can be brought to a stop at 303 by removing the Motive force atsuch a time that the material comes to rest at 304. PID(Proportional-Integrating-differential) methods and/or controllers arevery effective for optimizing fluid delivery accounting for hysteresiseffects within the fluid transfer mechanisms and the microlumens.

In FIG. 3B, material can be applied at 302 and 302′ and thenindividually advanced as described above, or may be advanced in tandemby the application of pressure differential across both fluidssimultaneously to yield the combined mixture 306 at the union of the twomicrolumens.

FIG. 3C is constructed by inducing the material assembled in acrystallization bolus 306 to crystallize. Pressure can then be appliedto the interior of the microlumen 301 to force the crystal containingbolus 307 along the microlumen 301 to the intersection 303. Thispressure can be applied hydraulically to port 302 or 302′, while sealingthe other, or to both ports 302 and 302′ simultaneously. The hydraulicpressure can be applied directly via syringe or syringe pump or via ahydraulic transfer fluid such as water or mineral oil using a fluidfilled syringe or syringe pump, with or without a connecting manifold tofacilitate the application of the hydraulic pressure to the ports 302and 302′. At harvest, by modulating the pressure difference between twooutlet ports, the unwanted crystallization liquor can be preferentiallyforced into the waste passage as bolus 308′, while concentrating thecrystal in a desired amount of crystallization liquor in bolus 308′. Thepressure can be modulated by differential pumping of two syringe pumpsconnected to the respective outlet ports. This can be done under manualcontrol with a simple joystick controller, or it can be accomplishedwith computer vision software, such as that provided by Keyance.

The methods and the devices of the present invention will now bedescribed with regard to the following figures.

FIGS. 4A-4C provide several embodiments of performing crystallizationswithin a lumen. It should be recognized that depending upon thediffering surface energies of the solutions and the enclosure the actualinterfaces may be convex, concave, flat or with elements of all three.For illustrative purposes, the interfaces are shown to be spherical.

FIG. 4A illustrates a crystallization mixture 401 formed within a lumen403 positioned between two dividers 400, 402. The lumen 403 is formed atleast partially by a substrate 407 and enclosed therein.

Generally, dividers may be used according to the present invention toseparate aliquots of material within the microvolumes, typically thelumens. The separated aliquots of materials may correspond to separateexperiments such as the crystallization trials described herein.

The dividers 400, 402 may be semi-permeable gases or liquids,semi-permeable gels or permeable gels, or thixotropic liquids, orimmiscible and impermeable liquids or beads. As a result, the interfaceformed between a crystallization and a divider may be a liquid/liquid,liquid/gas interface, liquid/solid or liquid/sol-gel interface. In someinstances, the interface may also be a membrane, gel, frit, or matrix tomodulate or alter the diffusion characteristics.

The dividers can be impermeable, semi-permeable or permeable. Forexample, semi-permeable substances such as air, oil, solvent, gel andbeads can be used as dividers. The dividers can also be physicalconstructions, such as a narrow pore, a thin passage, a frit or sinteredbeads or powders.

In one variation, the dividers function to modulate diffusioncharacteristics between adjacent samples. For example, the one or moredividers may be formed of a semi-permeable material that allowsdiffusion between adjacent crystallization samples.

In crystallizations performed within lumens according to the presentinvention, there may be one or multiple crystallization conditions,either related or unrelated in a given lumen. The dividers serve toseparate and optionally isolate the different crystallizationconditions. For example, a second crystallization condition, potentiallyone of many, is illustrated by dividers 404, 406 surroundingcrystallization 405. The dividers and the gap 403 may optionally beomitted.

Alternatively, the substance to be crystallized can be element 401, with400 and 402 being crystallization agents, either identical or different.In this instance, element 403 functions as a barrier between onecondition and the next. As illustrated in FIG. 4B, element 403 can beomitted for a series of crystallizations.

By positioning barrier material on opposing sides of a crystallizationwithin the microlumen, the crystallization may be encased and its lengththereby controlled. Examples of barrier materials that may be usedinclude, but are not limited to immiscible solvents or solids. Thebarrier materials may form a complete or partial barrier. Completebarriers prevent the crystallization from traversing the barriermaterial. Partial barriers limit the rate at which components of thecrystallization traversing the barrier material. Examples of partialbarrier materials include, but are not limited to polymers or solventsthat allow for diffusion. Diffusion within the crystal conditions can befurther modified by the use of thixotropic agents, gels or sols toprevent convective movements of the solutions.

As an alternative method, the substance to be crystallized may beelements 400, 402, 404, and 406. In this instance, elements 401 and 405may be crystallizing agents. Element 403 meanwhile may be a barrier orcan be another crystallization agent.

In all cases, the crystallization agent can be mixed prior to attemptingto perform the crystallization within the lumen or can act in situ, withno prior mixing.

FIG. 4B illustrates a crystallization performed within a lumen wheremultiple crystallization conditions are simultaneously employed. Asillustrated, elements 411, 413, and 415 can be crystallizationconditions, either premixed with crystallization agents or not. Ifelements 411, 413, and 415 are premixed, then elements 410, 412, 414,416 may optionally be a semi-permeable gas or liquid, a semi-permeablegel or permeable gel, a thixotropic liquid, an immiscible liquid, animpermeable liquid or bead, or a crystallization agent.

In the instance where 411, 413, and 415 are not premixed, thenminimally, 412 and 414 are crystallization agents and the termini, 410and 416 are a barrier (e.g., either a bead, an impermeable substance ora gas bubble).

In the instance that the crystallization is rapid, it is not necessaryto have impermeable termini. Instead, diffusion from the termini can beused as an additional crystallization agent.

FIG. 4C illustrates a crystallization performed within a lumen where aseries of crystallization agents are set up for crystallization againsta series of substances to be crystallized. In FIG. 4C, thecrystallization agents are shown as elements 420, 422, 424, and 426. Thecrystallization attempts comprising substances to be crystallized areshown as elements 421, 423, and 425. These crystallization attempts mayor may not be identical.

The sequential crystallizations can be formed in the microlumen by thesequential addition of the materials in inverse order. Thus, sample 406may be loaded into the microlumen, followed by 405, followed by 404 andso forth. Obviously, the microlumen can be loaded from right to left orleft to right. The individual crystallizations may be made on thecassette by using a manifold such as the one show in FIG. 3D, and thenvarying the relative pressures in the manifold individually or inparallel to achieve the desired mixing. For instance, barrier materialmight be loaded via 309, protein via 309′, semi-permeable material via309″, and a crystallization agent via 309′″. The alternating volumes offluid can be easily made outside of the microlumen by the sequentialloading of a syringe pump. To do this, the syringe loads the firstsample volume from the source of the first material 400 by creating apressure differential. The second material 401 is then loaded by thesame method. Then the next material 403 is loaded until either thevolume limit is reached upon the syringe or the desired contents of themicrolumen have been loaded. The syringe pump can then unload thecontents into the inlet port on the microlumen.

FIG. 4C might be conveniently constructed through the use of a Tee asshown in FIG. 3A, wherein a series of crystallization conditions couldbe injected into the microlumen, alternating with suitable injections ofmaterial to be crystallized. It will be appreciated by those skilled inthe art, that complete droplets can be made by small bursts ofdifferential pressure.

FIGS. 5A-5D illustrate crystallizations being performed within lumenswhere one or more of the elements of the crystallization experimentchange along a length of the lumen. As will be explained, the change canoccur discretely or continuously, and need not be changed in a simplelinear method.

FIG. 5A illustrates a lumen 501 where the crystallization condition isdifferent across the lumen.

FIG. 5B illustrates a series of substances to be crystallized, shown aselements 510, 511, 512, and 514. These substances are present in asingle gradient 513 such that the different elements are exposed todifferent crystallization conditions.

FIG. 5C illustrates an alternative to the embodiment shown in FIG. 5B.In this embodiment, a series of different crystallization agents 520,521, 522, and 524 are present within the lumen and are used to providedifferent conditions for crystallizing substance 523 present across thelumen.

FIG. 5D illustrates diffusion between the various elements in an in situcrystallization. Termini 1 and 7 share a single interface for diffusion.Each of the remaining portions of the in situ crystallization share atleast two distinct interfaces for diffusion. Thus, a single substance tobe crystallized, present across the lumen, can be assayed against two ormore crystallization agents simultaneously. For example, substance 2 isshown to share two separate interfaces, which can cause crystals to groweither near the 1 to 2 interface or the 2 to 3 interface. Crystalsgrowing in the center of 2 are indicative of a substance that requiresaspects of both 1 and 3 to crystallize.

The gradient shown in FIG. 5A can be created by using a “Tee” shown inFIG. 3A together with a series of mixing baffles downstream. Initiallyall of the input flow comes from one of the ports, for example 302″. Theflow in the second port 302′″ is increased, usually with a correspondingdecrease in the amount of material flowing in at 302″. The relativeinjection volumes, the total volume injected and the rate at which theychange will affect the final gradient produced. Gradients may be formedoff chip by similar means. The addition of a series of crystallizationagents can be effected via the use of a “Tee” as described above, or maybe individually loaded in an inlet port.

FIG. 6A illustrates a crystallization performed within a lumen where asingle crystallization condition 601 occupies an entire crystallizationspace.

FIG. 6B illustrates multiple crystallizations being performed within alumen where dividers 611, 612, 615, 617 are used between thecrystallizations 610, 612, 614, 616, and 618. The dividers are shown inthe figure to have planar surfaces adjacent the crystallizations.

FIG. 6C illustrates multiple crystallizations being performed within alumen where dividers 621, 623, 625 are used between the crystallizations622, 624. The dividers are shown in the figure to have curved, convexsurfaces adjacent the crystallizations 622, and 624 that havecomplementary concave surfaces. The actual shape of the meniscusdividing the samples is a function of the surface tension at theinterface and the surface of the microlumen.

FIG. 7A shows a device for performing a series of crystallizationswithin a series of lumens where each lumen comprises a loading port 701and an unloading port 703 and a lumen body 702 interconnecting theports.

FIG. 7B shows a cross section of a device 700 for performing acrystallization within a lumen where the lumen 702 is not enclosed.

FIG. 7C shows a cross section of a device 700 for performing acrystallization within a lumen where the lumens 702 are rectangular inshape.

FIG. 7D shows a cross section of a device 700 for performing acrystallization within a lumen where the lumens 702 are curved ortubular in shape.

FIG. 7E shows a device for performing crystallizations within a seriesof lumens where the lumens are loaded with samples 705, 707 that areseparated by divider or modifier segments 704, 706, 708. It should beappreciated that each discrete sample may have conditions that arepotentially unique and unrelated to adjacent samples. The dividers ormodifiers positioned between the samples can be permeable,semi-permeable or impermeable.

The series of crystallizations shown in 7E can be created via the samemethods used to create samples 4A above. To expedite the process, it ispreferable to load some or all of the channels simultaneously.

FIG. 8A shows a device 800 for performing a series of differentcrystallizations within a series of lumens where each lumen comprises aloading port 801 and unloading port 803 and a lumen body 802interconnecting the ports.

FIG. 8B illustrates a single lumen 802 in which a barrier 805 isadjacent to the crystallization condition 806 bounded by second barrier807. The crystallization conditions can be a larger volume, or the samevolume or smaller volume than the barriers. A more complex form of FIG.8B, is shown in FIG. 8C. It is noted that the barriers used hereincorrespond to the dividers described above.

FIG. 8C illustrates a diffusion crystallization. The barrier 805′, whichcan be either permeable or impermeable, is adjacent to thecrystallization condition 806′, which is bounded by the barrier 807′,which can be either permeable, semi-permeable and is adjacent tocrystallization condition 808′, which differs from condition 806′ in atleast one component. Condition 808′ is bounded by boundary condition809′, which can be permeable, semi-permeable or impermeable. Conditions806′ and 808 form a set of linked crystallization conditions, whose rateof equilibration is modulated by the properties of barrier 807′. Thisexample can be easily generalized to an entire crystallization channelor plate by suitable construction of the conditions and the plateitself. This is illustrated with regard to FIG. 8D.

FIG. 8D illustrates a multi-component crystallization being performed ina single lumen. The multi-component crystallization consists of endbarriers 809 and 839 and crystallization conditions 811 through 837,each separated from its adjacent neighbor conditions by a permeable orsemi-permeable barrier 810, repeating along the channel as 810′ betweeneach condition 811 through 837. Any number of conditions can be coupledvia semi-permeable or permeable barriers depending on the dimensions ofthe lumen, the design of the plate and crystal arrays and the volumes ofthe various crystal conditions.

Various methods may be performed using devices that allow for diffusionbetween adjoining flows within a single lumen. For example, in oneembodiment, a microfluidic method is provided that comprises: deliveringfirst and second fluids to a lumen of a microfluidic device such thatthe first and second fluids flow adjacent to each other within the lumenwithout mixing except for diffusion at an interface between the firstand second fluids, wherein the first fluid is different than the secondfluid. In a variation according to this embodiment, the composition ofat least one of the first and second fluids is varied over time as it isdelivered to the lumen so that the fluid forms a gradient with regard toa concentration of at least one component of the fluid that changesalong a length of the lumen. In another variation according to thisembodiment, the microfluidic device may comprise a plurality of lumens,the method further comprising delivering first and second fluids to eachof the plurality of lumens.

According to this embodiment, the same first and second fluids may bedelivered to each of the plurality of lumens. Alternatively, differentfirst and second fluids are delivered to the different lumens of theplurality of lumens. The first and second fluids may also have a same ordifferent flow rate within the lumen.

In another embodiment, a microfluidic method is provided that comprises:delivering first and second fluids to a lumen of a microfluidic devicesuch that the first and second fluids flow adjacent to each other withinthe lumen without mixing except for diffusion at an interface betweenthe first and second fluids, wherein the first fluid is different thanthe second fluid and a composition of at least one of the first andsecond fluids delivered to the lumen is varied so that the compositionof at least one of the first and second fluids within the lumen variesalong a length of the lumen.

In yet another embodiment, a microfluidic method is provided thatcomprises: delivering first, second and third fluids to a lumen of amicrofluidic device such that the first, second and third fluids flowadjacent to each other within the lumen without mixing except fordiffusion at an interface between the first, second and third fluids,wherein the first, second and third fluids are different than each otherand a composition of at least one of the first, second and third fluidsdelivered to the lumen is varied so that the composition of at least oneof the first, second, and third fluids within the lumen varies along alength of the lumen.

According to any of these embodiments, the composition of at least oneof the first, second and third fluids may be varied over time as it isdelivered to the lumen so that the fluid forms a gradient with regard toa concentration of at least one component of the fluid that changesalong a length of the lumen. Also according to any of these embodiments,the microfluidic device may comprise a plurality of lumens, the methodcomprising delivering first, second and third fluids to each of theplurality of lumens. The same or different first, second and thirdfluids may be delivered to each of the plurality of lumens. Optionally,at least one of the first, second and third fluids have a different flowrate than another of the fluids within the lumen. Also, at least one ofthe first, second and third fluids may have the same flow rate thananother of the fluids within the lumen. Also according to any of theseembodiments, the first, second and third fluids may be combined to formdifferent crystallization conditions for crystallizing a molecule suchas a protein. In one variation, the first, second and third fluidscombine to form different crystallization conditions, the second fluidcomprising the material to be crystallized and being positioned betweenthe first and third fluids.

In regard to any of these embodiments, dividers may optionally be usedin one or more of the first, second and optionally third or more fluids.These dividers may be used to set up multiple separate aliquots in thefluid flow where the dividers are positioned.

One particular application of these various methods is the use thefirst, second and optionally third or more fluids to form differentcrystallization conditions for crystallizing a material such as aprotein.

Devices and methods that allow for diffusion between adjoining flowswithin a single lumen will now be described in regard to FIGS. 9A-9D.

FIG. 9A shows an embodiment of a device 900 being used to perform aseries of different crystallizations within a series of multi-lumenassemblies 901, 901′ where each multi-lumen assembly comprises at leastone and preferably two loading 902, 903 and unloading 907, 908 ports anda lumen body 901 interconnecting the ports.

The fluids for each crystallization may be contained in two distinctfluid flows 902, 903 from the port that are in contact with each otheralong a shared interface 906.

It is noted that this shared interface 906 is not a structure but is aninterface that forms between the two distinct fluid flows as a result oflaminar flow within a microvolume dimensioned lumen. By contrast, FIGS.10A-10B describe adjacent fluid flows in separate lumens where apermeable or semi-permeable shared wall is positioned between the lumensthat allows for diffusion between fluids in the separate lumens.

The fluid flows consist of a crystallization condition 909 and a seriesof crystallization conditions 904 and 904′ that are separated by abarrier 905, which may be permeable, semi-permeable or impermeable. Thisarrangement enables the simultaneous examination of many differentconditions against a single condition.

The lumen shown in FIG. 9A can be either preloaded with a fluid or not.It is preferable, however, that the pairs of fluid flows besimultaneously loaded via the inlet ports 902 and 903. Having anexisting fluid in the lumen may facilitate maintaining the laminar flownecessary to maintain a uniform interface 906 between the two fluidflows.

A method for loading a single channel has been described above. Thisprocess can be used to produce the samples introduced via inlet port902. Simultaneous with the injection of the material via port 902 is theinjection of the desired material 909. This method can be easilygeneralized to more than two fluid flows.

FIGS. 9B-9D show an embodiment of a crystallization experiment that maybe performed using the device of FIG. 9A.

FIG. 9B illustrates an enlargement of a lumen of the device shown inFIG. 9A which illustrates some of the different simultaneous diffusionsthat are made possible by the invention. In fluid flow 910, aliquots ofcrystallization agents 912, 914 are positioned on opposing sides ofpermeable or semi-permeable barrier 916. Further aliquots may bepositioned upstream and downstream of the portion of the flow shown.Barriers 918, 920 on the outer sides of aliquots 912, 914 may beimpermeable and thereby isolate these aliquots relative to the remainderof the fluid flow. As shown, barriers 918, 920 are permeable orsemipermeable, allowing for diffusion further along fluid flow 910.

As illustrated in FIG. 9C, when the intervening barriers are permeableor semi-permeable, diffusion occurs through the barrier to form agradient from condition 912 to condition 914. Furthermore, fluid flow922 adjacent fluid flow 910 also forms a diffusion front between the twoflows. As a result, diffusion between fluid flow 922 and aliquots 912,914 also occurs. These different diffusions are illustrated in FIG. 9Cby circles 924, 926, 928 and 930.

As can be seen from FIG. 9C, this method provides for the diffusion ofcrystallization components longitudinally between differing conditionsin a manner that can be regulated through the suitable choice ofbarriers as well as laterally across an interface formed by the laminarflow of microfluid flows.

FIG. 9D illustrates crystals forming at different locations. Forexample, crystal 932 is shown positioned between the fluid flows andcrystal 934 is shown positioned within divider 916. Meanwhile, crystal936 is shown to be formed on the portion of the diffusion interfacefarthest from aliquot 914. The positioning of the crystals relative tothe aliquots and the fluid flows can be used to indicate whichcrystallization conditions are conducive and not conducive to crystalformation. As can be seen, the multiplicity of different conditions thatmay be formed by using these multiple different diffusion fronts allowsa great level of diversity of crystallization conditions to be created.

Crystallization conditions can be examined as part of a time series andthe time and location of nucleation, initial crystallization orprecipitation can be observed or derived. By using known, or observeddiffusion rates, the actual conditions at the nucleation orcrystallization points can be determined and used for further, moredetailed crystallizations. This method thus allows for a finer and morecomplete examination of crystallization conditions than can be affordedby single condition mixing of crystallization agents and the material tobe crystallized.

The diffusions illustrated in FIGS. 9B-9D are based on diffusion betweenfirst and second adjacent fluid flows. It should be recognized that thismethod and device design can be readily extended to three, four, or moreadjacent fluid flows.

The diffusions illustrated in FIGS. 9B-9D are also based on multiplealiquots in the first fluid flow. These multiple aliquots may have thesame or different composition. They may comprise crystallization agentsand/or the material to be crystallized It should be recognized that thesecond fluid flow may also have multiple aliquots.

Various devices and methods are also provided that allow for diffusionbetween adjoining lumens. For example, in one embodiment, a microfluidicdevice is provided that comprises: a substrate; a first lumen at leastpartially defined by the substrate; and a second lumen; wherein thefirst and second lumens share a common wall with each other that allowsfor diffusion between the two lumens over at least a portion of thelength of the two lumens.

In another embodiment, a microfluidic device is provided that comprises:a substrate; a plurality of sets of lumens, each set comprising a firstlumen at least partially defined by the substrate, and a second lumen,wherein the first and second lumens share a common wall with each otherthat allows for diffusion between the two lumens over at least a portionof the length of the two lumens.

It is desirable for these devices to allow for a high degree of parallelexperimentations. Accordingly, the devices preferably comprise at least4, 8, 12, 24, 96, 200, 1000 or more sets of lumens with adjoining walls.

According to each embodiment, the common wall may optionally comprise amembrane, gel, frit, or matrix that allows for diffusion between the twolumens.

Also according to each embodiment, the device may further comprise athird lumen, the third lumen sharing a common wall with at least one ofthe first and second lumens so as to allow for diffusion between thelumens over at least a portion of the length of the lumens.

Microfluidic methods are also provided. For example, in one embodiment,a microfluidic method is provided comprising: delivering a first fluidto a first lumen of a microfluidic device and a second, different fluidto a second lumen of the microfluidic device, the first and secondlumens sharing a common wall that allows for diffusion between thelumens over at least a portion of the length of the lumens; and havingthe first and second fluids diffuse between the first and second lumens.

It is noted in regard to the devices and methods that laminar flowallows for separate fluid flows to be delivered in a same lumen, asdiscussed above in regard to FIGS. 9A-9D. As a result, it should also berecognized that the common wall need not be present along an entirelength of the adjacent lumens. Further, it should be noted that one, twoor more separate fluid flows may be added to each lumen.

Devices and methods that allow for diffusion between adjoining lumenswill now be described in regard to FIGS. 10A-10B

FIG. 10A illustrates an embodiment of a device 1000 being used toperform a series of different crystallizations within a series ofmulti-lumen assemblies 1001, 1001′ where each multi-lumen assemblycomprises a first lumen 1002 and a second lumen 1004, the first andsecond lumens sharing a common wall 1006 that allows for diffusionbetween the lumens over at least a portion of the length of the lumens.Each lumen has a separate loading 1008, 1010 and unloading 1012, 1014ports that are each in fluid communication with a lumen.

A separate fluid flow 1016, 1018 is delivered to each lumen through theloading ports 1008, 1010.

It is noted that the common wall 1006 in FIG. 10A differs from theshared interface 906 of FIG. 9A because it is an actual permeable orsemi-permeable structure positioned between the lumens that allows fordiffusion between fluid in the separate lumens.

FIG. 10B illustrates an alternate embodiment where the multi-lumenassembly comprises a first lumen 1002, a second lumen 1004, and a thirdlumen 1005 where the first, second and third lumens share common wall1006, 1007 that allows for diffusion between the lumens over at least aportion of the length of the lumens.

It is noted that a composition of at least one of the first and secondfluids may be varied so that the composition of at least one of thefirst and second fluids varies along a length of the lumen. Thecomposition of at least one of the first and second fluids may also varyover time as it is delivered to the lumen so that the fluid forms agradient with regard to a concentration of at least one component of thefluid that changes along a length of the lumen. Depending on theexperiment, the same or different first and second fluids may bedelivered to each of the plurality of first and second lumens.

It is also noted that the first and second fluids may have a same ordifferent flow rate within the lumen.

When a third separate lumen is provided, the method may optionallyfurther comprise delivering a third fluid to a third lumen which sharesa common wall with at least one of the first and second lumens, thecommon wall allowing for diffusion between the third lumen and the firstor second lumen over at least a portion of the length of the lumens.

It should be recognized that the embodiments of FIGS. 9A-9D and FIGS.10A-10B may optionally be combined. For example, a device according toFIG. 10A or 10B may be employed where two or more fluids flows areintroduced into a single lumen, as illustrated in FIGS. 9A-9D.

FIGS. 10C-10E show an embodiment of a crystallization experiment thatmay be performed. Shown in the figures are an enlargement of the doublelumen device shown in FIG. 10A. Lumen 1002 is separated from lumen 1004by the permeable or semi-permeable wall 1006.

In FIG. 10C, lumen 1002 is illustrated as containing a series ofcrystallization agents 1020, 1020′, and 1020″. These crystallizationagents in lumen 1002 are separated by impermeable dividers 1022′ and asemi-permeable divider 1022. This allows separate aliquot to be createdin that lumen. It should be recognized that the dividers employed inthis embodiment are optional.

Lumen 1004 meanwhile is shown to contain the mixture to be assayed forcrystallization, such as a protein solution in a buffer. Once the lumensare filled, diffusion between the differing chemical mixtures begins.

As shown in FIG. 10D, after some time has passed, diffusion can occurboth through the semi-permeable internal divider 1022, as well asthrough the permeable or semi-permeable wall 1006. The chemicalgradients from the crystallization agents are illustrated as diffusionfronts 1024, 1026, diffusing into the crystallization mixtures, and asthe intra-lumen diffusion from 1021. It is noted that diffusion front1021 can also permeate through the permeable or semi-permeable wall1006, resulting in a joint diffusion front 1025.

FIG. 10E illustrates a sample series of crystal growths that haveoccurred after some diffusion has occurred. Crystal 1028 is in thecenter of diffusion front 1024 and has resulted largely from the actionof crystallization condition 1020′. Crystal 1029 has grown on thediffusion interface 1025 and is therefore indicative of a crystal thatneeds chemical moieties of both condition 1020′ and condition 1020″ toform. In contrast, crystal 1030 has formed on the portion of thediffusion interface farthest from condition 1020′, suggesting that someaspect of condition 1020′ slows or prevents crystal growth in thecontext of the crystallization agent 1020″. As can be seen, themultiplicity of different conditions that may be formed by using thesemultiple different diffusion fronts allows a great level of diversity ofcrystallization conditions to be created.

As shown in FIG. 10F, multiple lumens 1002, and 1004 may be separated bypermeable or semi-permeable wall 1006. Lumens 1004 and 1005 areseparated by permeable or semi-permeable wall 1007. By the suitableloading of crystallization conditions, 1040, 1041, 1042, 1043, 1044,1045, 1046, 1047, 1048, and 1049, the skilled artisan can produce manywell defined opportunities for diffusion between the various conditions.By the design of suitable experiments the diffusion rates of thechemical moieties within crystallization conditions can be determined.Once the diffusion patterns have been established, the location ofcrystals within the lumen can be used to interpolate the nucleation andcrystal growth conditions between the existing conditions.

FIG. 11A illustrates a single lumen with integral mixing and harvestingchannels. The single channel comprises an inlet assembly of at least twoinlet ports 1102, 1103 and mixing channel 1104, a crystallizationchannel 1101 and a harvesting assembly 1107, comprised of at least oneharvesting port 1105 and preferably two ports 1106 for harvesting.

FIG. 11B shows an embodiment of a device 1100 for performing a series ofdifferent crystallizations within a series of lumens 1101, 1108-1114where each lumen comprises integral mixing and harvesting channels.Conditions with each channel may consist of identical conditions, or ofmultiple crystallization agents or of multiple substances to becrystallized or any combination thereof.

The “Y” shown in FIGS. 11A and 11B is easily utilized to alternate aseries of materials. Syringes or syringe pumps can alternately delivermaterial to ports 1102 and 1103. Simply, a small interruptible vacuumcan be applied to either 1105 or 1106 and the other can be sealed.Alternatively, the vacuum can be applied to both. Whenever a sample isloaded into 1102, port 1103 is sealed and the vacuum is applied at1105/1106 to transfer the appropriate volume into the “Y” 1104. When thedesired volume has been transferred, the pressure differential isremoved. Material can then be loaded at 1103, port 1102 is then sealedand a pressure difference sufficient to deliver the required volume into1104 is applied. The process can then be repeated. For ease of control,it may be preferable to preload the lumens with a hydraulic transferfluid. Similarly, it is preferable to apply constant pressure differencebetween the pair 1102/1103 and the pair 1105/1106. The lumen 1101 canthen be loaded by alternating the supply of material from 1102 and thesupply of material from 1103.

FIG. 12A illustrates a device 1200 comprising a series of lumens, eachlumen having attached to it an array of individual crystallization cells1204, each cell having at least one separate inlet 1202 or outlet 1203and at least one channel connecting the cell to the lumen 1201. Eachcrystallization cell may have an exclusive inlet and outlet, giving anarray of independent cells, or the cells may be linked, or multiplexedwith a common inlet or outlet lumen 1201, which can have a single 1202or multiple ports 1202, 1205. Substances unique to each crystallizationcell are loaded via the port 1203, with the excess being drawn off viathe common lumen. Substances common to all cells in a sub-array 1208,consisting of port 1202, manifold 1201, port 1205 and all thus linkedcrystallization cells and ports, can be loaded through ports 1202 and1205 by any combination of injection of suction via the ports within thesub-array. By suitable application of driving forces, substances can bedriven into any one of the attached crystallization cells, either inparallel or individually.

FIG. 12B illustrates an embodiment of an individual crystallization cellshown in FIG. 12A. If the device is to consist of individually accessedcrystallization cells, then port 1201 is unique to each cell. If thecassette includes multiple sub-arrays, then lumen 1201 may be commonwith the other crystallization cells of the sub-array.

FIG. 12C illustrates an embodiment of an individual crystallization cell1204 shown in FIG. 12A where the cell comprises a crystallization agent1207 and a substance 1206 to be crystallized. The exact nature of themeniscus between the substances is highly dependent upon both thesequence of addition of the crystallization materials, their relativevolume and the surface properties of the supporting materials of thecassette, e.g. surface energy, hydrophobicity, hydrophilicity, oradsorbed materials.

4. Delivery of Materials to Microsized Lumens

Materials may be added to the devices of the present invention by avariety of different methods and mechanisms. For example, material maybe added to a given lumen by the sequential addition of the volumes ofmaterials that need to be added or may be delivered as a single bolus.Commercial robots such as the Staubli can deliver small volumes ofmaterial with the high degree of accuracy needed to repeatedly deliverthe necessary drops into entry ports of the lumens. For improvedaccuracy, multiple deliveries can be used to create the final, largervolume, from a series of smaller volumes.

Volume of materials can be delivered by a number of differentmechanisms, such as ultrasonic dispensers, peristaltic pumps, syringes,syringe pumps or stepper motor driven plungers. Materials can bedelivered to multiple different lumens individually, in parallel withina channel, or in parallel across the entire device. For someembodiments, it may be desirable to deliver two or three conditionssimultaneously for optimal loading. Alternatively, it is possible to usepin arrays to deliver the fluid.

The device may also be docked with a manifold in order to delivermaterials to the lumens of the device. This manifold can be mated withat least one or multiple inlet ports. The channels in the chip can thenbe filled individually, or in parallel from the manifold. The fillingcycle may be entirely in parallel, or the filling cycle may involvemultiple docking events. If the device is docked multiple times todifferent manifolds, the materials can be added by alternately matingthe device with, for example, a protein manifold, a barrier manifold,and a crystallization manifold. The materials may be pressure driveninto the device, or may be applied with vacuum, or a combinationthereof. Under some constructs, it may be advantageous to pre-fill thedevice with a fluid. This fluid can then be displaced by the pressureaddition of material, or this fluid may be removed actively be anapplied vacuum, or a combination thereof to deliver the necessaryfluids. Pre-filling the device has advantages in the fluidics, and alsofor the alteration or modulation of the surface properties of the lumen.

5. Transport in Microfluidic Devices Using Centrifugal Force

One feature of the present invention relates to the use of centrifugalforce to cause material to flow within the microlumens of devicesaccording to the present invention. Through the use of centrifugalforce, fluids can be loaded, measured, filtered, mixed and incubatedwithin a lumen. The centrifugal force serves to generate hydrostaticpressure to drive the fluids through the lumens, reservoirs, filters andmanifolds. This method has the advantages of speed, tightly enclosedfluids to minimize evaporation, and simplicity since there are no movingparts on the device to break or become fouled through the application ofexternal energies. The use of centrifugal force is compatible with awide variety of fluids.

Optionally, the centrifugal forces are applied such that at least 0.01g, 0.1, 1 g, 10 g, 100 g or more force is applied to the material in thedevice to cause the material to move within the microvolumes.

Applying the centrifugal forces may be performed by rotating the device.Optionally, the centrifugal forces are applied by rotating the device atleast 10 rpm, 50 rpm, 100 rpm or more. It is noted that the rotationalaxis about which the microfluidic device is rotated may be positionedwithin or outside the lateral footprint of the microfluidic device.

A particular advantage of the use of centrifugal force is the ability tomake hundreds to thousands to hundreds of thousands of replicate volumemeasurements simultaneously. Accordingly, devices may be designed sothat material in at least 4, 8, 12, 36, 96, 200, 1000 or more differentmicrovolumes are transported when centrifugal force is applied.

In addition, since a common amount of force can be applied to each lumenand the shapes of the lumens can be closely controlled, replicate volumemeasurements can be made with a high degree of reproducibility. Forexample, devices can be designed so that the volume of fluid deliveredfrom in a given microvolume upon the application of centrifugal force iswithin 50%, 25%, 10%, 5%, 2%, 1% or less of the volume of fluiddelivered in any other microvolumes.

A further feature of the use of a device employing centrifugal force isthe ability to preload crystallization agents. This can be used todramatically enhance the speed and efficiency of the crystallizationsetup.

In one embodiment, a microfluidic method is provided that comprises:taking a microfluidic device comprising a plurality of microvolumes; andcausing movement of material in a same manner within the plurality ofmicrovolumes by applying centrifugal forces to the material.

In another embodiment, a microfluidic method is provided that comprises:taking a plurality of microfluidic devices, each device comprising aplurality of microvolumes; and causing movement of material in a samemanner within the plurality of microvolumes of the plurality of devicesby applying centrifugal forces to the material.

In a variation, the plurality of microfluidic devices may be stackedrelative to each other when the centrifugal forces are applied. Theplurality of microfluidic devices may also be positioned about arotational axis about which the plurality of microfluidic devices arerotated to apply the centrifugal forces.

In another embodiment, a microfluidic method is provided that comprises:taking a microfluidic device comprising a plurality of microvolumes; andphysically moving the device so as to effect a same movement of materialwithin the plurality of microvolumes. Physically moving the devicepreferably causes centrifugal force to be applied, for example, byrotation of the device about an axis. According to this embodiment, thematerial moved in each of the plurality of microvolumes by movement ofthe device preferably has a same quantity.

In another embodiment, a microfluidic method is provided that comprises:taking a microfluidic device comprising a plurality of microvolumes; andaccelerating or decelerating a motion of the device so as to effect asame movement of material within the plurality of microvolumes.According to this embodiment, the motion of the device is optionally arotation of the device. In such instances, acceleration or decelerationmay be caused by a change in a rate of rotation of the device.

In another embodiment, a microfluidic device is provided that comprises:a substrate; and a plurality of microvolumes at least partially definedby the substrate, each microvolume comprising a first submicrovolume anda second submicrovolume that is in fluid communication with the firstsubmicrovolume when the device is rotated, the plurality of microvolumesbeing arranged in the device such that fluid in the firstsubmicrovolumes of multiple of the microvolumes are transported tosecond submicrovolumes of the associated microvolumes when the device isrotated.

In yet another embodiment, a microfluidic device is provided thatcomprises: a substrate shaped so as to provide the device with an axisof rotation about which the device may be rotated; and a plurality ofmicrovolumes at least partially defined by the substrate, eachmicrovolume comprising a first submicrovolume and a secondsubmicrovolume that is in fluid communication with the firstsubmicrovolume when the device is rotated, the plurality of microvolumesbeing arranged in the device such that fluid in the firstsubmicrovolumes of multiple of the microvolumes are transported to thesecond submicrovolumes of the associated microvolumes when the device isrotated about the rotational axis. Optionally, the second microvolumesare lumens.

The device may optionally comprise a mechanism that facilitates thedevice being rotated about the rotational axis. For example, thesubstrate may define a groove or hole at the rotational axis thatfacilitates the device being rotated about the rotational axis.Optionally, a center of mass of the device is at the rotational axis andthe substrate defines a groove or hole at the rotational axis thatfacilitates the device being rotated about the rotational axis. In onevariation, the device is disc shaped, the substrate defining a groove orhole at the rotational axis of the disc that facilitates the devicebeing rotated about the rotational axis.

In another embodiment, a microfluidic method is provided that comprises:taking a microfluidic device comprising a substrate, and a plurality ofmicrovolumes at least partially defined by the substrate, eachmicrovolume comprising a first submicrovolume and a secondsubmicrovolume where the first submicrovolume and second microvolume arein fluid communication with each other when the device is rotated;adding fluid to a plurality of the first submicrovolumes; and rotatingthe device to cause fluid from the plurality of first submicrovolumes tobe transferred to the second submicrovolumes in fluid communication withthe first submicrovolumes.

In another embodiment, a microfluidic method is provided that comprises:taking a plurality of microfluidic devices, each comprising a substrate,and a plurality of microvolumes at least partially defined by thesubstrate, each sample microvolume comprising a first submicrovolume anda second submicrovolume where the first submicrovolume and secondsubmicrovolume are in fluid communication with each other when thedevice is rotated; adding fluid to a plurality of the firstsubmicrovolumes in the plurality of microfluidic devices; and rotatingthe plurality of microfluidic devices at the same time to cause fluidfrom the plurality of first submicrovolumes to be transferred to thesecond submicrovolumes in fluid communication with the firstsubmicrovolumes.

In another embodiment, a microfluidic method is provided that comprises:taking a microfluidic device comprising a substrate, and a plurality ofmicrovolumes at least partially defined by the substrate, eachmicrovolume comprising a first and a second submicrovolume where thefirst and second submicrovolumes are in fluid communication with eachother when the device is rotated about a rotational axis of the device;adding fluid to a plurality of the first submicrovolumes; and rotatingthe device about the rotational axis of the device to cause fluid in thefirst submicrovolumes to be transferred to the second submicrovolumes.

In another embodiment, a microfluidic device is provided that comprises:a substrate; one or more microvolumes at least partially defined by thesubstrate, each microvolume comprising a first submicrovolume, a secondsubmicrovolume where fluid in the first submicrovolume is transported tothe second submicrovolume when the device is rotated about a firstrotational axis, and a third submicrovolume where fluid in the firstsubmicrovolume is transported to the third submicrovolume when thedevice is rotated about a second, different rotational axis.

In another embodiment, a microfluidic device comprising: a substrate;one or more microvolumes extending along a plane of the substrate, eachmicrovolume comprising a first submicrovolume, a second submicrovolumewhere fluid in the first submicrovolume is transported to the secondsubmicrovolume when the device is rotated about a first rotational axisthat is positioned further away from the second submicrovolume than thefirst submicrovolume, and a third submicrovolume where fluid in thefirst submicrovolume is transported to the third submicrovolume when thedevice is rotated about a second, different rotational axis that ispositioned further away from the third submicrovolume than the firstsubmicrovolume.

In another embodiment, a microfluidic method is provided that comprises:taking a microfluidic device comprising a substrate and a plurality ofmicrovolumes at least partially defined by the substrate, eachmicrovolume comprising an first submicrovolume, a second submicrovolumewhere fluid in the first submicrovolume is transported to the secondsubmicrovolume when the device is rotated about a first rotational axis,and a third submicrovolume where fluid in the first submicrovolume istransported to the third submicrovolume when the device is rotated abouta second, different rotational axis; adding fluid to the firstsubmicrovolumes of the microvolumes; and in any order rotating thedevice about the first and second rotational axes to cause fluid fromthe first submicrovolumes to be transferred to the second and thirdsubmicrovolumes.

In another embodiment, a microfluidic device is provided that comprises:a substrate; and a plurality of microvolumes at least partially definedby the substrate, each microvolume comprising a first submicrovolume anda second submicrovolume in fluid communication with the firstsubmicrovolume when the device is rotated about a first rotational axis,wherein rotation of the device about the first rotational axis causes afixed volume to be transported to each of the second submicrovolumes.

According to this embodiment, the plurality of microvolumes mayoptionally further comprise one or more outlet submicrovolumes in fluidcommunication with the first submicrovolume.

Also according to this embodiment, the plurality of microvolumes mayoptionally further comprise one or more outlet submicrovolumes wherefluid in the first submicrovolume not transported to the secondsubmicrovolume when the device is rotated about a first rotational axisis transported to one or more one or more outlet submicrovolumes whenthe device is rotated about a second, different rotational axis.

In another embodiment, a microfluidic device is provided that comprises:a substrate; a first microvolume at least partially defined by thesubstrate comprising a first submicrovolume; a second submicrovolumewhere fluid in the first submicrovolume is transported to the secondsubmicrovolume when the device is rotated about a first rotational axis;and a second microvolume at least partially defined by the substratecomprising a third submicrovolume; a fourth submicrovolume where fluidin the third submicrovolume is transported to the fourth submicrovolumewhen the device is rotated about the first rotational axis; and whereinfluid in the second and fourth submicrovolumes are transported to afifth submicrovolume where the second and fourth submicrovolumes aremixed when the device is rotated about a second, different rotationalaxis.

According to this embodiment, the fifth submicrovolume may optionally bein fluid communication with the second and fourth submicrovolumes viathe first and third submicrovolumes respectively.

Also according to this embodiment, the device may further comprise oneor more outlet submicrovolumes in fluid communication with the first andthird submicrovolumes.

Also according to this embodiment, the device may further comprise oneor more outlet submicrovolumes in fluid communication with the first andsecond submicrovolumes where fluid in the first and thirdsubmicrovolumes not transported to the second and fourth submicrovolumeswhen the device is rotated about the first rotational axis istransported to one or more one or more outlet submicrovolumes when thedevice is rotated about a third, different rotational axis.

In yet another embodiment, a microfluidic method is provided thatcomprises: taking a microfluidic device comprising a substrate, and aplurality of microvolumes at least partially defined by the substrate,each microvolume comprising a first submicrovolume and a secondsubmicrovolume in fluid communication with the first submicrovolume;adding fluids to the first submicrovolumes; and applying a centrifugalforce to the device to cause a same volume of fluid to be transported tothe second microvolumes from the first submicrovolumes.

Optionally, the microvolumes may further comprise an outletsubmicrovolume in fluid communication with the first submicrovolumes. Insuch instances, the method may further comprise transporting fluid inthe first submicrovolume to the outlet submicrovolume that was nottransported to the second submicrovolume when the centrifugal force wasapplied. The method may also further comprise transporting fluid in thefirst submicrovolume to the outlet submicrovolume that was nottransported to the second submicrovolume when the device is rotatedabout a first rotational axis by rotating the device about a second,different rotational axis.

In another embodiment, a microfluidic method is provided that comprises:taking a microfluidic device comprising a substrate, a first microvolumeat least partially defined by the substrate comprising a firstsubmicrovolume and a second submicrovolume where fluid in the firstsubmicrovolume is transported to the second submicrovolume when thedevice is rotated about a first rotational axis, and a secondmicrovolume at least partially defined by the substrate comprising athird submicrovolume and a fourth submicrovolume where fluid in thethird submicrovolume is transported to the fourth submicrovolume whenthe device is rotated about the first rotational axis, the microvolumesfurther comprising a fifth submicrovolume where fluid in the second andfourth submicrovolumes are mixed when the device is rotated about asecond, different rotational axis; adding a first fluid to the firstsubmicrovolume and a second fluid to the third submicrovolume; rotatingthe device about the first rotational axis to transport the first andsecond fluids to the second and fourth submicrovolumes; and rotating thedevice about the second rotational axis to transport the first andsecond fluids from the second and fourth submicrovolumes to the fifthsubmicrovolume.

In one variation, the fifth submicrovolume is in fluid communicationwith the second and fourth submicrovolumes via the first and thirdsubmicrovolumes respectively.

Optionally, the method further comprises removing fluid from the firstand third submicrovolumes that is not transported to the second andfourth submicrovolumes prior to rotating the device about the secondrotational axis.

More specific examples of devices and methods according to thesenumerous embodiments will now be described in relation to the figures.

FIG. 13A illustrates a device for forming crystallizations by rotationof the device, thereby applying centrifugal force. The device 1300comprises multiple crystallization wells 1301, each having at least oneinlet port 1302, a crystallization channel 1303 and an outlet port 1304.It is understood that during centrifugation, the radially outermost portwill, due to centrifugal forces be the outlet port. However, for thepurposes of loading the cassette, either port 1302, 1304 may be used asan inlet or outlet port. The device can have a centering device 1305 tocenter the device during centrifugation, or alternatively, the devicemay be inserted in a receiver designed to mate with the device.

As can be seen, the device is similar in design to a compact disc,comprising a flat, circular plate of substrate with a hole in themiddle, preferably at the center of mass of the device. Incorporatedinto the substrate is an array of crystallization chambers. This designallows the crystallization agents to be added to the device. Then, whenthe device is rotated, the crystallization agents in the differentchambers are each caused to enter a corresponding crystallization well.

Given the symmetry of the design and the uniformity of the centrifugalforce that is applied, the design of the device provides for a compactsystem where crystallization agents can be first added and stored in thedevice. Then, when the device is ready to be used, the device can berotated to cause the prior added crystallization agents to move withinthe device. As illustrated in FIG. 13A, the rotational axis about whichthe microfluidic device is rotated may be within a lateral footprint ofthe device.

The design of the device also allows for multiple devices to be stackedupon each other. This allows for a great number of devices to beprocessed in parallel. FIG. 13B illustrates a plurality of the devicesshown in FIG. 13A where the devices are stacked relative to each otherwhen the centrifugal forces are applied so that the same forces areapplied to all of the devices.

FIG. 14A illustrates another embodiment of a device 1400 that isdesigned to move fluids within the device by centrifugal force. Thisdesign allows for the precise measurement of very small volumes withoutthe use of moving parts, electromotive force or active pumps within thedevice. The device consists of at least two inlet chambers 1401, 1401′,a measurement channel 1402 for each inlet, a waste channel 1403 fromeach inlet 1401 to a waste reservoir 1404 or outlet, a mixing manifold1405 connecting the measurement channels 1402, 1402′ and thecrystallization chamber 1406. The manifold, can encompass the inletport, or by pass it. The measurement channels can be of identical ordiffering volumes, dependent upon the need. The crystallization chambercan be of any shape, shown here as either circular 1406 or rectangular1406′. Only one waste channel is illustrated, but each measurementchannel has an associated waste channel. These channels can beindependent, or by suitable design can form a manifold.

The device may be employed as follows; into each inlet chamber 1401, avolume of crystallization agent or substance to be crystallized isadded. The volume that is added does not need to be precise or accurate.Instead, it is sufficient that the volume is greater than a minimumvolume for the measurement channel 1402. The crystallization agents canbe dispensed in advance of the substance to be crystallized, enablingthe device to be made in advance and used as needed. Once the inletchambers are all filled, the device is rotated with the centrifugalforce vector approximately aligned as shown, for the loading spin (A).

The centrifugal force fills the measurement channel 1402 completely,leaving some residue in the inlet chamber 1401. A subsequent measurementspin (B), removes the excess from the inlet chamber and deposits theexcess in the waste reservoir 1404 or port, leaving the inlet chamberempty. At this point, the device may be stored until needed. The inletports may be sealed by the application of a tape, lid, septum, or bystacking the devices together.

The rotational axis about which the microfluidic device illustrated inFIG. 14A is rotated is positioned further away from the measurementchannel 1402 than the inlet chamber 1401. This provides the centrifugalforce vector the directionality that is shown during the loading spin(A). The rotational axis about which the microfluidic device is rotatedmay be within or outside of the lateral footprint of the device.

The substance to be crystallized is then added into each inlet chamber1401′. The volume does not need to be precise or accurate. Instead, itis sufficient that the volume be greater than a certain minimum for themeasurement channel 1402′. Once the inlet chambers are all filled, thedevice is centrifuged with the centrifugal force vector as shown, forthe loading spin (A). This fills the measurement channel 1402′completely, leaving some residue in the inlet chamber 1401′.

A subsequent measurement spin (B) with the centrifugal force vectorapproximately in this direction, removes the excess from the inletchamber and deposits the excess in the waste reservoir 1404 or port,leaving the inlet chamber empty. At this point, the device may again bestored until needed. The inlet ports may be sealed by the application ofa tape, lid, septum, or by stacking the plates together.

It is noted that the rotational axis about which the microfluidic deviceis rotated in the subsequent measurement spin (B) is positioned in adifferent location than the rotational axis during the load spin (A). Inthis instance, the different rotational axes are laterally offsetrelative to each other. The rotational axis for the measurement spin (B)is also positioned further away from the waste reservoir 1404 or portthan the inlet chamber 1401. This positioning causes the centrifugalforce vector to be in the direction illustrated in regard to themeasurement spin (B). It is noted that the rotational axis about whichthe microfluidic device is rotated during the measurement spin may alsobe within or outside of the lateral footprint of the device. Althoughthe rotational axes are shown to be parallel and laterally offsetrelative to each other, it should be recognized that the axes may alsobe angled relative to each other.

Crystallization, or the test of crystallization is initiated bycentrifugation with the centrifugal force vector approximately in thedirection of the crystallization initiation spin (C). This drives thecrystallization agent or agents and the substance to be crystallizedthrough a mixing manifold 1405 into a crystallization chamber 1406.

It is noted that the rotational axis about which the microfluidic deviceis rotated during the crystallization initiation spin (C) is positionedin a different location than during the load spin (A) or the measurementspin (B). For example, the rotational axis for the crystallizationinitiation spin (C) is positioned further away from the inlet chamber1401 than the measurement channel 1402. This positioning causes thecentrifugal force vector to be in the direction illustrated in regard tothe load spin (C), which in this case is in the opposite direction thanthe centrifugal force vector for the load spin (A). It is again notedthat the rotational axis about which the microfluidic device is rotatedduring the load spin may also be within or outside of the lateralfootprint of the device.

The process illustrated in FIG. 14A is an example of a microfluidicmethod that is provided by the present invention that comprises: takinga microfluidic device comprising a substrate and a plurality ofmicrovolumes at least partially defined by the substrate, eachmicrovolume comprising an first submicrovolume, a second submicrovolumewhere fluid in the first submicrovolume is transported to the secondsubmicrovolume when the device is rotated about a first rotational axis,and a third submicrovolume where fluid in the first submicrovolume istransported to the third submicrovolume when the device is rotated abouta second, different rotational axis; adding fluid to the firstsubmicrovolumes of the microvolumes; and in any order rotating thedevice about the first and second rotational axes to cause fluid fromthe first submicrovolumes to be transferred to the second and thirdsubmicrovolumes.

FIG. 14B illustrates how multiple devices, such as the device shown inFIG. 14A may be processed together. As illustrated, the multiple devicesmay be positioned radially about a rotational axis. During eachdifferent load, measurement, and initiation spins, each device may bepositioned relative to the rotational axis so that the correspondingvector is extending radially away from the rotational axis. It should benoted that the multiple devices may alternatively or in addition bestacked relative to each other, as illustrated in FIG. 13B.

It is noted in regard to the various embodiments involving centrifugalforce, such as the embodiment shown in FIG. 14A, that acceleration anddeceleration, created by a change in a rate of rotation of the device,can be used. In particular, when a device is rotating and the rate ofchange of rotation of the device is zero or close to zero, then theprimary component of the force vector is radial. However, when thedevice is initially at a constant rotational speed, which could be zero,and then as the rotational speed of the device is increased, the primarycomponent is initially orthogonal to the radius, and in the rotationalplane, tangential to the rotation. This is also true if the device isdecelerated. It is also noted that the faster the device is accelerated,the larger the magnitude of the force.

This fact can be used to modulate the flow of liquid within themicrofluidic device. A large tangential force vector as the device isbeing accelerated causes the liquid within device to initially beginflowing in the counter to the direction of the initial force. Thus, theinertial response of the fluid to the centripetal acceleration is toappear to lag the acceleration of the device. Similarly, if the deviceis decelerated, the fluid will lag the deceleration.

This enables the production of devices that have different fluidbehavior depending upon the direction of rotation and the rate ofacceleration or deceleration.

In one example, described in relation to the device illustrated in FIG.14A, if the initial loading spin has a rotational axis on a side of thedevice adjacent the crystallization chamber 1406 and the device israpidly accelerated clockwise, fluid placed in loading port 1401 willflow into chamber 1402. Conversely, if the device is initially rotatedcounter-clockwise with a rapid acceleration, the initial force upon theliquid will direct the liquid largely toward port 1403 and hence to thewaste chamber 1404. If the device is accelerated slowly, the primarycomponent of the force vector acting upon the liquid will be radial andthe liquid will flow from the loading port 1401 into the chamber 1402,regardless of the direction of rotation. Skilled artisans willappreciate that by suitable design of the substrate and by manipulationof the rate of change of the rotational speed, the fluid flow in thedevice can be modulated to achieve very different outcomes. It will beunderstood by those skilled in the art that when the centrifugal forceis neither parallel to nor normal to the plane of the device, additionalasymmetries in fluid flow may be exploited for more complex fluidpartitioning and combinations.

FIG. 15A illustrates how centrifugal force can be used to performprecise measurements. FIG. 15A illustrates a repeating unit of thecentrifugal array in more detail. In this embodiment, one inlet forcrystallization agents 1501 and one inlet for a substance to becrystallized 1501′ are shown. Note that the lumens 1502, 1502′connecting to the measurement lumens have a short neck near the inletchamber, orthogonal to the measurement spin. V represents the measuredvolume after the measurement spin. During the measurement spin, excessmaterial in the inlet chamber, and the excess above V is centrifugallyejected through lumens 1503, 1503′ and hence through lumens 1504, 1504′to the exit port or reservoir. Note that lumens 1503, 1503′, also have anarrow neck, initially oriented parallel to and in the oppositedirection to the loading spin vector, ensuring that the liquids proceeddown 1502 or 1502′ to the measurement lumens.

FIG. 15A thus illustrates a microfluidic method that comprises: taking amicrofluidic device comprising a substrate, and a plurality ofmicrovolumes at least partially defined by the substrate, eachmicrovolume comprising a first submicrovolume and a secondsubmicrovolume in fluid communication with the first submicrovolume;adding fluids to the first submicrovolumes; and applying a centrifugalforce to the device to cause a same volume of fluid to be transported tothe second microvolumes from the first submicrovolumes.

FIG. 15A thus also illustrates an embodiment of a microfluidic devicethat comprises: a substrate; and a plurality of microvolumes at leastpartially defined by the substrate, each microvolume comprising a firstsubmicrovolume and a second submicrovolume in fluid communication withthe first submicrovolume when the device is rotated about a firstrotational axis, wherein rotation of the device about the firstrotational axis causes a fixed volume to be transported to each of thesecond submicrovolumes.

As illustrated, the plurality of microvolumes may optionally furthercomprise one or more outlet submicrovolumes where fluid in the firstsubmicrovolume not transported to the second submicrovolume when thedevice is rotated about a first rotational axis is transported to one ormore one or more outlet submicrovolumes when the device is rotated abouta second, different rotational axis. When the microvolumes furthercomprise an outlet submicrovolume in fluid communication with the firstsubmicrovolumes, the method may further comprise transporting fluid inthe first submicrovolume to the outlet submicrovolume that was nottransported to the second submicrovolume when the centrifugal force wasapplied. The method may also further comprise transporting fluid in thefirst submicrovolume to the outlet submicrovolume that was nottransported to the second submicrovolume when the device is rotatedabout a first rotational axis by rotating the device about a second,different rotational axis.

FIGS. 15B-15G illustrate how centrifugal force can be used to performprecise measurements and mixing. More specifically, these figuresillustrate a microfluidic device that comprises: a substrate; a firstmicrovolume at least partially defined by the substrate comprising afirst submicrovolume; a second submicrovolume where fluid in the firstsubmicrovolume is transported to the second submicrovolume when thedevice is rotated about a first rotational axis; and a secondmicrovolume at least partially defined by the substrate comprising athird submicrovolume; a fourth submicrovolume where fluid in the thirdsubmicrovolume is transported to the fourth submicrovolume when thedevice is rotated about the first rotational axis; and wherein fluid inthe second and fourth submicrovolumes are transported to a fifthsubmicrovolume where the second and fourth submicrovolumes are mixedwhen the device is rotated about a second, different rotational axis. Aswill be illustrated, the fifth submicrovolume may optionally be in fluidcommunication with the second and fourth submicrovolumes via the firstand third submicrovolumes respectively. As will also be illustrated, thedevice may further comprise one or more outlet submicrovolumes in fluidcommunication with the first and third submicrovolumes. As will also beillustrated, the device may further comprise one or more outletsubmicrovolumes in fluid communication with the first and secondsubmicrovolumes where fluid in the first and third submicrovolumes nottransported to the second and fourth submicrovolumes when the device isrotated about the first rotational axis is transported to one or moreone or more outlet submicrovolumes when the device is rotated about athird, different rotational axis.

FIGS. 15B-15G also illustrate a method that comprises: taking amicrofluidic device comprising a substrate, a first microvolume at leastpartially defined by the substrate comprising a first submicrovolume anda second submicrovolume where fluid in the first submicrovolume istransported to the second submicrovolume when the device is rotatedabout a first rotational axis, and a second microvolume at leastpartially defined by the substrate comprising a third submicrovolume anda fourth submicrovolume where fluid in the third submicrovolume istransported to the fourth submicrovolume when the device is rotatedabout the first rotational axis, the microvolumes further comprising afifth submicrovolume where fluid in the second and fourthsubmicrovolumes are mixed when the device is rotated about a second,different rotational axis; adding a first fluid to the firstsubmicrovolume and a second fluid to the third submicrovolume; rotatingthe device about the first rotational axis to transport the first andsecond fluids to the second and fourth submicrovolumes; and rotating thedevice about the second rotational axis to transport the first andsecond fluids from the second and fourth submicrovolumes to the fifthsubmicrovolume.

It is noted that different sets and subsets of combinations describedherein can be performed without departing from the present invention.

Referring to FIG. 15B a device is shown where a crystallization agent1507 has been added into the entry port, or well 1501. Also shown is thematerial to be crystallized 1507′ in a second entry port or well. Thesematerials need not be added contemporaneously.

FIG. 15C illustrates the effect of centrifugal force on the samples thatwere loaded to the device in FIG. 15B. Upon the application ofcentrifugal force, the bulk of the material 1507 and 1507′ proceeds tofill the respective measurement lumens 1502 and 1502′. This leaves someamount of excess material 1508 and 1508′ in the initial loading wells1501 and 1501′, respectively.

In FIG. 15D, the centrifugal force vector is changed such that the forcenow directs the excess crystallization agent and excess material to becrystallized 1508 and 1508′ toward the waste ports 1504, 1504′ via therespective waste lumens 1503, 1503′. After applying this centrifugalforce, the measurement channel is filled with material to the point V inevery measurement lumen.

FIG. 15E shows each lumen filled to point V, resulting in precise volumemeasurements.

In FIG. 15F, the centrifugal force vector is again changed to align inthe direction shown. Centrifugal force in this direction drives thecrystallization agent 1507 and the material to be crystallized 1507′from the measurement lumens 1502, 1502′, across the inlet ports 1501,1501′ and through a manifold 1505, 1505′ to the mixing manifold 1506 andhence into the crystallization chamber 1500, as the mixed material 1509.

FIG. 15G illustrates the final result, where the crystallization chamber1500 has been filled with the combination of the material to becrystallized and the crystallization agent, or agents.

As will be appreciated, the process of making precise microfluidicmeasurements and precise mixing by using centrifugal force can beperformed in a highly parallel manner, both by incorporating numerousmicrovolumes into a given device, and by applying centrifugal force tomultiple different devices at the same time, wherein the variations inacceleration, or deceleration, will be uniformly applied over alldevices and all lumens within said devices.

6. Use of the Devices of the Present Invention to Determine CrystalGrowth Conditions

One of the intended uses of the devices of the present invention is forimproving the process of discovering novel crystal growth conditions. Byusing the devices of the present invention, a simultaneous, multiplefactor approach can be implemented.

Current methods of vapor diffusion, hanging drop, sitting drop anddialysis evaluate a single test condition in each instance. By contrast,the present invention allows for multiple different crystallizationconditions to be created in the same lumen, thereby allowing formultiple different crystallization conditions to be tested. In someembodiments, gradients are created which create the multiple differentcrystallization conditions. Diffusion of either the sample beingevaluated and/or the enclosing medium having a viscosity such that thediffusion of the chemical moieties for crystallization is much fasterthan the diffusion of bulk material allows for the gradients to becreated. This can be achieved either through intrinsically viscousmaterials or additives such as agarose, acrylamide, silica gel, or PEG,or by the use of filter plugs, or by the use of enclosing channels thatare sufficiently thin in at least one dimension to limit macroscopicflow such that diffusion of the chemical moieties for crystallizationdominate. These samples can be affected by sample droplets in a channel,droplets within an enclosing crystallization medium, or crystallizationdroplets or islands within an enclosing volume of sample.

While the present invention is disclosed with reference to preferredembodiments and examples detailed above, it is to be understood thatthese examples are intended in an illustrative rather than limitingsense, as it is contemplated that modifications will readily occur tothose skilled in the art, which modifications will be within the spiritof the invention and the scope of the appended claims. The patents,papers, and books cited in this application are to be incorporatedherein in their entirety.

1. A method for determining crystallization conditions for a material,the method comprising: taking a microfluidic device comprising one ormore lumens having microvolume dimensions and a plurality of differentcrystallization samples within at least one of the lumens, the pluralityof different crystallization samples being separated from each otherwithin the lumen by one or more dividers, the plurality ofcrystallization samples comprising a material to be crystallized andcrystallization conditions that vary among the plurality ofcrystallization samples; transporting the plurality of differentcrystallization samples within the lumens; and identifying a precipitateor crystal formed in the one or more lumens.
 2. The method according toclaim 1 wherein transporting the plurality of different crystallizationsamples within the one or more lumens is performed by a method selectedfrom the group consisting of electrophoresis, electroosmotic flow andphysical pumping.
 3. The method according to claim 1 whereintransporting the plurality of different crystallization samples withinthe one or more lumens is performed by electrokinetic materialtransport.
 4. The method according to claim 1 wherein the material to becrystallized is a macromolecule.
 5. The method according to claim 1wherein the material to be crystallized is a protein.
 6. The methodaccording to claim 1 wherein the material to be crystallized is amacromolecule with a molecular weight of at least 500 daltons.
 7. Themethod according to claim 1 wherein the material to be crystallized isselected from the group consisting of viruses, proteins, peptides,nucleosides, nucleotides, ribonucleic acids, deoxyribonucleic acids. 8.The method according to claim 1 wherein the material to be crystallizedcontains at least two or more materials selected from the groupconsisting of viruses, proteins, peptides, nucleosides, nucleotides,ribonucleic acids, deoxyribonucleic acids, small molecules, drugs,putative drugs, inorganic compounds, metal salts, organometalliccompounds and elements.
 9. The method according to claim 1 wherein theone or more lumens have a cross sectional diameter of less than 2.5 mm.10. The method according to claim 1 wherein the one or more lumens havea cross sectional diameter of less than 1 mm.
 11. The method accordingto claim 1 wherein the one or more lumens have a cross sectionaldiameter of less than 500 microns.
 12. A method according to claim 1,wherein the one or more divider are formed of an impermeable material.13. A method according to claim 12, wherein the impermeable material isan impermeable liquid.
 14. A method according to claim 12, wherein theimpermeable material is an impermeable solid.
 15. A method according toclaim 1, wherein the one or more dividers are formed of a permeablematerial.
 16. A method according to claim 1, wherein the one or moredividers are formed of a semimpermeable material.
 17. A method accordingto claim 16, wherein the semipermeable material is a gas.
 18. A methodaccording to claim 16, wherein the semipermeable material is a liquid.19. A method according to claim 16, wherein the semipermeable materialis a gel.
 20. A method according to claim 16, wherein the one or moredivders form an interface selected from the group consisting ofliquid/liquid, liquid/gas interface, liquid/solid and liquid/sol-gelinterface.
 21. A method according to claim 1, wherein the one or moredividers are selected from the group consisting of a membrane, gel,frit, and matrix.
 22. A method according to claim 1, wherein the one ormore dividers function to modulate diffusion characteristics betweenadjacent crystallization samples.
 23. A method according to claim 1,wherein the one or more dividers are formed of a semipermeable materialthat allows diffusion between adjacent crystallization samples.
 24. Amethod according to claim 1, further including forming the plurality ofdifferent crystallization samples within the one or more lumens.
 25. Amethod for determining crystallization conditions for a material, themethod comprising: taking a microfluidic device comprising one or morelumens having microvolume dimensions and a plurality of differentcrystallization samples within the one or more lumens, the plurality ofcrystallization samples comprising a material to be crystallized andcrystallization conditions that vary among the plurality ofcrystallization samples; transporting the plurality of differentcrystallization samples within the one or more lumens; identifying aprecipitate or crystal formed in the one or more lumens; and performinga spectroscopic analysis on the identified precipitate or crystal whilewithin the lumen.
 26. A method according to claim 25, wherein thespectroscopic analysis is selected from the group consisting of Raman,UV/VIS, IR, and x-ray spectroscopy.
 27. A method according to claim 25,wherein the spectroscopic analysis is x-ray spectroscopy.
 28. A methodaccording to claim 27, wherein the x-ray spectroscopy is x-raydiffraction.
 29. A method according to claim 25, further includingforming the plurality of different crystallization samples within theone or more lumens.
 30. A method according to claim 25, wherein aplurality of crystallization samples are comprised in at least onelumen.
 31. A method according to claim 25, wherein transporting theplurality of different crystallization samples within the one or morelumens is performed by eletrokinetic. material transport.
 32. A methodaccording to claim 25, wherein transporting the plurality of differentcrystallization samples within the one or more lumens is performed by amethod selected from the group consisting of electrophoresis,electroosmotic flow and physical pumping.